ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 253

Stress, Drugs and Neuroscience

Neurobiological Effects of Social Stressors and DrugExposure in Young and Adolescent Rats

LINNEA GRANHOLM

ISSN 1651-6192ISBN 978-91-513-0282-9urn:nbn:se:uu:diva-346151

Dissertation presented at Uppsala University to be publicly examined in A1:107a,Biomedicinskt centrum, Husargatan 3, Uppsala, Wednesday, 9 May 2018 at 13:00 for thedegree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conductedin English. Faculty examiner: Associate Professor Annika Thorsell (Linköpings University,Department of Clinical and Experimental Medicine).

AbstractGranholm, L. 2018. Stress, Drugs and Neuroscience. Neurobiological Effects of SocialStressors and Drug Exposure in Young and Adolescent Rats. Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Pharmacy 253. 79 pp. Uppsala: ActaUniversitatis Upsaliensis. ISBN 978-91-513-0282-9.

Experiences early in life or during adolescence modulate neuronal networks in the immaturebrain and consequently lay the foundation for future susceptibility or resilience towardspsychiatric disorders. The objective in this thesis is to understand, in part, how the surroundingenvironment shapes the brain of a young individual. Three types of negative life events werestudied, in an animal model, for their effects on the brain reward system (i.e., endogenousopioids and dopamine) and voluntary drug intake. These were: disruption of maternalcare, disruption of interaction with peers, and exposure to drugs. Stress, in the form ofmaternal separation, altered expression of opioid genes in the dorsal striatum and amygdala,and the response to subsequent alcohol intake on these genes was dependent on early lifeconditions. Basal levels of endogenous opioids were also dependent on how the animals werehoused in early adolescence. Short single housing (30 minutes) caused an acute stress responseas evidenced by increased serum corticosterone and nociceptin/orphanin FQ in brain areasassociated with stress. A prolonged single housing resulted in a marked decrease of Met-Enk-Arg6-Phe7 (i.e., a marker of enkephalins) in several brain areas. The endogenous opioids werealso affected by repeated exposure of ethanol during adolescence; ethanol intoxication increasedthe accumbal levels of Met-Enk-Arg6-Phe7 and decreased those of β-endorphin. Residual effectsof the adolescent ethanol exposure were found in Met-Enk-Arg6-Phe7 levels in the amygdala,ventral tegmental area, and substantia nigra. Furthermore, rats exposed to ethanol as adolescentshad alterations in the dopamine dynamics in the dorsal striatum. Both endogenous opioids anddopamine are essential in mediating rewarding properties. Alterations of these systems, causedby environmental disturbances and alcohol exposure, presented herein could explain, in part,the increased susceptibility for alcohol- and substance use disorders later in life.

Keywords: Adolescence, alcohol exposure, amphetamine, beta-endorphin, dopamine,dynorphin, early life, endogenous opioids, enkephalin, gene expression, high-speedchronoamperometry, nociceptin/orphanin FQ, radioimmunoassay, self-administration, socialstress, voluntary alcohol intake

Linnea Granholm, Department of Pharmaceutical Biosciences, Box 591, Uppsala University,SE-75124 Uppsala, Sweden.

© Linnea Granholm 2018

ISSN 1651-6192ISBN 978-91-513-0282-9urn:nbn:se:uu:diva-346151 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-346151)

Hörde fåglarna sjunga om vår

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I. Granholm, L., Todkar, A., Bergman, S., Nilsson, K., Comasco E., Nylander I. (2017) The expression of opioid genes in non-classical reward areas depends on early life conditions and ethanol intake. Brain Research, 1668:36–45

II. Granholm, L*., Roman, E*., Nylander, I. (2015) Single housing during adolescence causes time-, area-, and peptide-specific altera-tions in endogenous opioids of rat brain. British Journal of Pharma-cology, 172:606-614

III. Granholm, L., Segerström L., Nylander I. (2018) Episodic ethanol exposure in adolescent rats – Effects on endogenous opioid peptides. In manuscript.

IV. Granholm, L*., Rowley, S*., Ellgren, M., Segerström, L., Nylander, I. (2015) Impact of adolescent ethanol exposure and adult amphetamine self-administration on evoked striatal dopamine re-lease in male rats. Psychopharmacology, 232(24):4421-4431

*Authors contributed equally

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11Early life ................................................................................................... 11Adolescence .............................................................................................. 12Alcohol ..................................................................................................... 12

Consumption behavior ......................................................................... 12Adolescent drinking ............................................................................. 14

Dopamine .................................................................................................. 14Endogenous opioids .................................................................................. 15Social behavior and endogenous opioids .................................................. 17

Early life ............................................................................................... 17Adolescence ......................................................................................... 17

Central circuitries in the addiction cycle .................................................. 18Ethanol ...................................................................................................... 19

Effects on dopamine in dorsal and ventral striatum ............................. 20Effects on endogenous opioids ............................................................ 21

Amphetamine ............................................................................................ 22

Methodology ................................................................................................. 24Maternal separation – simulation of early life stress ........................... 24Experimental housing conditions – simulation of social environments .............................................................................................................. 25Alcohol exposure – simulation of adolescent binge-like drinking ...... 26Voluntary alcohol drinking – evaluation of consumption behavior .... 27Operant self-administration – evaluation of drug-taking behavior ...... 28In vivo high-speed chronoamperometry – analysis of dopamine ......... 29Real-time polymerase chain reaction – analysis of gene expression ... 31Radioimmunoassay – quantification of peptides ................................. 33

Aims .............................................................................................................. 35

Methods ......................................................................................................... 36Animals ..................................................................................................... 36Maternal separation (Paper I) ................................................................... 37Adolescent housing conditions (Paper II) ................................................ 38Adolescent ethanol exposure (Paper III-IV) ............................................. 38Adult voluntary ethanol intake (Paper I) .................................................. 38Operant self-administration (Paper IV) .................................................... 38

Operant boxes apparatus ...................................................................... 38Sucrose training ................................................................................... 39Intravenous amphetamine self-administration ..................................... 39

High speed chronoamperometry (Paper IV) ............................................. 40Quantitative polymerase chain reaction (Paper I) .................................... 41Tissue stabilization (Paper III) ................................................................. 41Radioimmunoassay (Paper II-III) ............................................................. 42Statistical analysis ..................................................................................... 42

Results and discussion ................................................................................... 44Early life stress, alcohol intake and opioid gene expression .................... 44

Amygdala ............................................................................................. 44Dorsal striatum ..................................................................................... 45

Adolescent housing conditions and opioid peptides ................................. 47Nociceptin/Orphanin FQ ...................................................................... 47Methionine-Enkephalin-Arginine6-Phenylalanine7 ............................. 49Dynorphin B ......................................................................................... 50

Adolescent alcohol exposure .................................................................... 50Effects of intoxication on opioid peptides ........................................... 51Residual effects on opioid peptides ..................................................... 52Residual effects on in vivo dopamine in the dorsal striatum ............... 53Subsequent amphetamine self-administration ..................................... 54Subsequent amphetamine self-administration and in vivo dopamine .. 56

Conclusions ................................................................................................... 57

Populärvetenskaplig sammanfattning ........................................................... 58

Acknowledgment .......................................................................................... 60

References ..................................................................................................... 62

Abbreviations

AFR Animal facility rearing ANOVA Analysis of variance cDNA Complementary DNA CRF Corticotrophin-releasing factor DAT Dopamine transporter DNA Deoxyribonucleic acid dPCR Digital polymerase chain reaction FR Fixed ratio GABA γ-aminobutyric acid HPA Hypothalamic-pituitary-adrenal MAO Monoaminooxidase MEAP Methionine-enkephalin-arginine6-phenylalanine7

mRNA Messenger ribonucleic acid MS15 Maternal separation 15 minutes MS360 Maternal separation 360 minutes N/OFQ Nociceptin/Orphanin FQ NOP Non-opioid receptor Oprd1 Opioid receptor delta type 1 Oprk1 Opioid receptor kappa type 1 Oprm1 Opioid receptor mu type 1 PCR Polymerase chain reaction PENK Proenkephalin PDYN Prodynorphin POMC Proopiomelanocortin PR Progressive ratio qPCR Quantitative real-time polymerase chain reaction RNA Ribonucleic acid VMAT2 Vesicular monoamine transporter-2 VTA Ventral tegmental area

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Introduction

Experiences in early life and adolescence modulate neuronal networks in the immature brain and lay the foundation for future susceptibility or resilience to psychiatric disorders. The objective in this thesis was to understand, in part, how the surrounding environment shapes the developing brain. Three types of negative life events were studied, in an animal model, for their ef-fects on the brain reward system (i.e., endogenous opioids and dopamine) and voluntary drug intake. These were; disruption of maternal care, disrup-tion of interaction with peers, and exposure to drugs.

In the last decades, this area of research has gained more attention. In 2010, the European IMAGEN consortium launched their plan of recruiting high school participants, to follow them throughout adolescence. The partic-ipants undergo a battery of tests ranging from psychological to neuroimaging and genomics to develop prevention strategies and improved therapies for mental health disorders. (Schumann et al., 2010). To answer similar ques-tions in a U.S population, the Adolescent Brain Cognitive Development study was created a couple of years later (Volkow et al., 2017).

Even if these large-scale clinical studies will provide a lot of knowledge, preclinical research is crucial when studying neurobiological consequences of adverse events in young individuals. Animal experimental models provide the opportunity for creating environments where the exposure of the stimuli can be controlled, and enable detailed brain analysis that cannot be made in humans for ethical reasons.

Early life The brain undergoes extensive maturation after birth. Both gray and white matter matures with synaptic formation, axonal growth, neuronal differentia-tion, neurotransmitter modulation and myelination (Dehaene-Lambertz and Spelke, 2015). Genes and environment affects brain development; hence, it is crucial for a child to grow up in an environment with limited stressors and external stimuli of an adverse nature.

From birth and throughout childhood, the most crucial relationship a child has is to its primary caregivers, usually the mother and/or father. A safe and stable environment is important to promote a secure attachment between caregiver and child, which in turn, is important for developmental outcomes.

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The caregivers' role is to act as a buffer from surrounding stressors. Mal-treatment in childhood, for example, parental neglect or abuse, alters trajec-tories of brain development (Teicher et al., 2016) and increases the risk of psychiatric disorders in adulthood (Carr et al., 2013). Alterations in neurobi-ology and behavior have also been found in preclinical models of early life stress, such as maternal separation (Lyons et al., 2010; Nylander and Roman, 2013).

Adolescence Adolescence is the period of transition from childhood to adulthood. Accord-ing to the World Health Organization, adolescence starts at age 10 and ends at 19 (WHO, 2014). This period also represents a time of extensive reorgani-zation and maturation of brain circuits involved in emotions, motivation, reward and cognition (Spear, 2013). These reflect the development of age-characteristic behaviors such as increased risk taking, impulsivity, and nov-elty seeking (Crews et al., 2007). These behaviors are not exclusive to hu-mans as they can be found in many species (Panksepp, 1981). Furthermore, the social roles and relationships undergo significant changes in adolescent humans (Heinrich and Gullone, 2006) as well as in rats (Spear, 2000). Al-most all mammalians develop an age-specific adolescent play behavior, which follows an inverted U-shaped pattern, i.e., the begins in early adoles-cence and declines with the approach of adulthood (Spinka et al., 2001). In rats, this behavior progressively increases between postnatal day 18-28 and peaks around 32-40, followed by a gradual decline (Panksepp, 1981). It is suggested that this type of social behavior is a part of the social and cogni-tive development as well as a preparation for forthcoming life events (Spinka et al., 2001). Preclinical research has shown that this type of behavior has reinforcing properties, and furthermore, it involves actions of many different neurotransmitters (e.g., opioids, dopamine, noradrenaline) (Trezza et al., 2014; 2010). Evidence from both human and experimental animal research shows that deprivation of social contact during adolescence can lead to se-vere consequences for normal neurobiological and behavioral maturation (Fone and Porkess, 2008; Heinrich and Gullone, 2006).

Alcohol Consumption behavior Alcohol drinking has a long history in human cultures. The word alcohol originates from the Arabic word “alkuhl” which means “something subtle”. The effects of alcohol consumption can range from the subtle to the more

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obvious, depending on the amount ingested. Alcohol (i.e., ethanol; alcohol and ethanol are used interchangeably in this thesis) is often consumed for its euphoric and sedative effects on the central nervous system. Intoxication also affects, for example, motor- and cognitive functions.

For the majority of alcohol consumers, the goal of drinking is to experi-ence the euphoric and positive reinforcing, and rewarding effects of intoxica-tion. Many individuals can handle repeated and intermittent drinking that sometimes leads to binge levels, i.e., 0.08 g/dl in about two hours according to National institute on Alcohol Abuse and Alcoholism (NIAAA). However, for others, binge drinking (i.e., five or more alcoholic drinks in males, or four or more alcoholic drinks in females, within a two hours (Spear, 2018)) can escalate into chronic use and addiction in which the goal of drinking shifts to finding relief from the negative effects of withdrawal (e.g., dyspho-ria and stress). The desire for becoming intoxicated increases, which leads to preoccupation with, and anticipation of, obtaining the drug. Finally, the per-son loses control of their drinking habit. This pattern is called the addiction cycle (Figure 1) and is further described in “The neuronal circuitries in the addiction cycle”. In this thesis, the effect of ethanol is not evaluated in ani-mals with addiction. Instead, it comprises studies of how the brain is affected in the early stages of alcohol use.

Figure 1. The circle represents the three stages in the addiction cycle—binge/intoxication, withdrawal/negative effects, and preoccupation/anticipation. Inside the circle is a schematic representation of a rat brain and the regions analyzed in the present thesis. The color-coding of the brain areas represents which stages in the addiction cycle that are most influenced by actions in this region; green, binge/intoxication; purple, withdrawal/negative effects; yellow, preoccupa-tion/anticipation; light gray, not defined.

Dorsal striatumNucleus accumbens

Ventral tegmental area

Amygdala

Hippocampus

Medial prefrontal cortex

Hypothalamus

Frontal cortex

Substantia nigra

Cingulate cortex

Pituitary

Binge/Intoxication

Pre

occu

patio

n/a

nticip

ation

Withdrawal/Negative effects

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Adolescent drinking Impulsivity and novelty seeking are predictors of drug use (Belin and Deroche-Gamonet, 2012; Jentsch et al., 2014). Not surprisingly, the debut of drug use usually occurs during adolescence, with alcohol being one of the most widely used drugs during this period. The consumption pattern is main-ly episodic binge drinking. Since the beginning of this century, there is a positive trend with decreasing alcohol consumption among Swedish teenag-ers, and the use is now down to historically low levels (CAN, 2017). How-ever, this annual report on adolescents drug habits in Sweden also investi-gated how adolescents experience their parents’ alcohol consumption. Ado-lescents growing up in a families with alcohol abuse use alcohol themselves more frequently and in greater quantities (CAN, 2017).

Both clinical and preclinical research shows that exposure to alcohol dur-ing adolescence affects neurobehavioral functions and brain maturation as well as intake of drugs (Guerri and Pascual, 2010; Hanson et al., 2011; Silveri et al., 2016; Spear, 2014; 2018; Squeglia et al., 2015). There is an association between the age of first alcohol use and an increased risk of sub-sequent alcohol and substance use disorders (DeWit et al., 2000; York et al., 2004). Spear (2015) argues that the increased liability with early drug use could be a result of three factors: (i) an environmental context in which drugs are frequently used; (ii) an inherited vulnerability for drugs of abuse, and (iii) a reshaping of the brain by drugs towards a susceptible state. Papers III and IV in this thesis focus on the third factor.

Dopamine In December 2000 the Swedish scientist Arvid Carlsson received the Nobel Prize for his achievements in the field of neurophysiology (Nobelprize.org) . He proved that dopamine is a neurotransmitter and not just a passive player in the synthesis of catecholamines (Carlsson et al., 1957).

A part of the synthesis of dopamine is outlined in Figure 3. The rate-limiting step is the conversion of tyrosine to dihydroxyphenylalanine (DO-PA). Dopamine is packed into synaptic vesicles by vesicular monoamine transporter-2 (VMAT2) before it is released in the synaptic cleft (Howell and Kimmel, 2008). Released dopamine acts on the dopamine receptors and thereafter it can be transported back into the presynapse by dopamine trans-porters (DAT) were it can be repacked into vesicles or metabolized by mon-oaminooxidase (MAO). Dopamine can also be metabolized outside the syn-apse by MAO or catechol-O-methyltransferase (i.e., COMT) (Meiser et al., 2013).

Dopamine receptors were discovered in 1978. They were divided into two classes, D1 or D2 receptors, on the basis of their ability to stimulate adenylyl

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cyclase (Garau et al., 1978; Spano et al., 1978). This classification is still valid today, but with gene cloning, three additional dopamine receptor sub-types (D3, D4 and D5) have been discovered (Grandy et al., 1991; Sokoloff et al., 1990; Sunahara et al., 1991; Van Tol et al., 1991). The D5 is consid-ered a D1-like receptor because of its similar transmembrane structure as the D1 receptor and as it shares the ability to stimulate adenylyl cyclase (Missale et al., 1998). The D3 and D4 receptors are classified as D2-like receptors because of their homology with the transmembrane domains of the D2 re-ceptor and as they do not stimulate adenylyl cyclase (Missale et al., 1998).

The Swedish researchers, Annika Dahlström and Kjell Fuxe, started map-ping the dopaminergic pathways in the central nervous system in the begin-ning of the 1960s (Dahlstrom and Fuxe, 1964) and their work laid the foun-dation for what we today know about the three major midbrain dopaminergic pathways: the nigrostriatal, mesolimbic and mesocortical (Björklund and Dunnett, 2007).

The dopamine receptors are activated in a variety of functions such as lo-comotor control, attention, cognition and regulation of food intake (Beaulieu and Gainetdinov, 2011). The focus in this thesis is on dopamine action in reward and reinforcement processes within the dorsal striatum.

Endogenous opioids The first references to use of opium are from the third century B.C. in the writings of Theophrastus (van Ree et al., 1999). The use of opioids – for both medical and recreational purposes – is well-documented throughout human history (van Ree et al., 1999), but the first proof of endogenous opi-oids came in the 1970s when it was shown that the brain contained specific binding sites for opiates (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). The hypothesis that there were multiple types of opioid receptors, i.e., μ, δ and κ, came from Martin and his co-workers (Martin et al., 1976), and the discovery of the opioid receptor ligand, enkephalin, (Hughes et al., 1975) confirmed their hypothesis. This was followed by the identification of two other endogenous opioid receptor ligands; endorphin and dynorphin (Cox et al., 1976; Goldstein et al., 1981). A fourth member of the opioid receptor family was discovered in the 1990s. This receptor was first called orphan receptor-like 1 or LC132 receptor (Bunzow et al., 1994; Mollereau et al., 1994), but the name was later changed to non-opioid recep-tor (NOP) when its endogenous ligand, nociceptin orphanin/FQ (N/OFQ) was discovered (Meunier et al., 1995; Reinscheid et al., 1995).

The opioid receptors belong to the G-protein coupled receptor family and exert their actions through inhibition of cyclic adenosine monophosphate (i.e., cAMP) via adenylyl cyclase and consequently decrease the neuronal activity by stimulating outward K+ and inhibiting voltage-gated Ca2+ chan-

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nels. μ-, δ- and κ-receptors are classical opioid receptors whereas the NOP receptor is a non-classical opioid receptor.

The endogenous ligands of the classical opioid receptors derive form three larger precursor proteins, proopiomelanocortin (POMC), proenkepha-lin (PENK) and prodynorphin (PDYN) that are cleaved by trypsin-like en-zymes to produce multiple bioactive peptides, see Figure 2 for details (Yaksh and Wallace, 2011). These ligands share an N-terminal amino acid sequence, Tyrosine-Glycine-Glycine-Phenylalanine-Methionine/Leucine, followed by various specific extensions of the C-terminus (Yaksh and Wallace, 2011). In contrast, N/OFQ does not share this signature sequence and it lacks affinity to the μ-, δ- and κ-receptors. As for the classical opioids, β-endorphin has strongest binding properties to μ- and δ-receptors, enkepha-lins have the highest affinity to δ-receptors but exert binding properties to μ-receptors as well, and dynorphins primarily target the κ-receptor (Yaksh and Wallace, 2011).

Figure 2. A simplified illustration of the endogenous opioid prohormones—proopiomelanocortin, proenkephalin, prodynorphin and proorphanin. Peptides in bold are those analyzed within this thesis. Abbreviations: ACTH, adrenocortico-tropic hormone; Arg, arginine; CLIP, corticotropin-like intermediate lobe peptide; Gly, glycine; Leu, leucine; Met, methionine; MSH, melanocyte-stimulating hor-mone; N/OFQ, nociceptin/orphanin FQ; Phe, phenylalanine. Adapted from (Yaksh and Wallace, 2011) and (Allen et al., 2001).

The overview of Le Merrer et al. (2009) shows the distribution patterns of receptors and precursor proteins for endorphins, dynorphins and enkephalins and reveals how extensively widespread the endogenous opioid system is in the central nervous system. This system is involved in a variety of physio-logical functions, which is easy to understand, since receptors, precursors

Proopiomelanocortin

Proenkephalin

Prodynorphin

Proorphanin

γ-MSH ACTH β-lipotropin

α-MSH CLIP γ-lipotropin β-endorphin

β-MSH

↓ ↓ ↓ ↓

↓

Met-enkephalin-Arg6Phe7

Met-enkephalin-Arg6Gly7Leu8

α-neoendorphin

β-neoendorphin

↓dynorphin 32

dynorphin A1-17 dynorphin B1-13

↓ ↓

dynorphin A1-8

↓

}}

Nocistatin N/OFQ

Met-enkephalin Leu-enkephalin

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and ligands are found in brain circuitries regulating, for example, reward, pain, and mood (Le Merrer et al., 2009; Lutz and Kieffer, 2013; Mansour et al., 1987; Yaksh and Wallace, 2011). The distribution of N/OFQ and NOP in the central nervous system includes brain areas (e.g., cortical regions, hippo-campus, amygdala, hypothalamus, locus coeruleus, nucleus accumbens, cau-date putamen) associated with mood regulation (including anxiety and stress), food intake, pain and locomotor activity (Mollereau and Mouledous, 2000; Witkin et al., 2014) .

Social behavior and endogenous opioids Early life As mentioned in “Early life”, the first bond an individual forms is to its pri-mary caregiver and the endogenous opioids have important roles in the neu-robiological underpinnings for parental bonding.

In humans and non-human primates, a variant of the gene encoding the μ-receptor influences attachment behavior and the relationship between parent and child (Barr et al., 2008; Copeland et al., 2011; Higham et al., 2011; Troisi et al., 2012). Furthermore, blocking μ-receptors in the early postpar-tum period reduces both caregiving and protective behavior in rhesus ma-caques (Martel et al., 1993; 1995). Stimulating μ-receptors with morphine increases foraging and hunting behavior in female rats at the cost of reduced maternal behavior (Bridges and Grimm, 1982; Felicio et al., 1991; Sukikara et al., 2007). Across species, administrations of μ-receptor agonists reduce infants’ distress calls when separated from their mothers, and opioid antago-nists induce or exacerbated distress behavior (Carden and Hofer, 1990a; 1990b; Herman and Panksepp, 1978; Kalin et al., 1988; Panksepp et al., 1980a). Furthermore, mice pups lacking the μ-receptor emit fewer distress calls when separated from their mother (Moles et al., 2004) In rodent models of parental neglect and loss, it has been shown that that maternal separation induces long-lasting alterations in endogenous opioids (Nylander and Roman, 2012) and alters the response to both naltrexone (Daoura and Nylander, 2011) and morphine (Kalinichev et al., 2001) in adulthood.

Adolescence Social grooming in non-human primates is fundamental for establishing long-term relationships with social support and protection (Dunbar, 2010). During this behavior, β-endorphins are released and grooming can be ma-nipulated with μ-opioid antagonists and agonists (Keverne et al., 1989; Martel et al., 1995; Meller et al., 1980; Schino and Troisi, 1992). Neuroim-aging data from humans shows that non-sexual, tactile contact stimulates the

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μ-opioid system, implicating that endogenous opioids are also important for our social bonding (Nummenmaa et al., 2016).

Social play behavior in rats involves a lot of physical contact such as wrestling, during which rodents emits positive ultrasonic vocalizations asso-ciated with joy (Vanderschuren et al., 2016). Endogenous opioids are re-leased during play (Panksepp and Bishop, 1981; Vanderschuren et al., 1995d) and thus contribute to the rewarding effects of the behavior (Calcagnetti and Schechter, 1992; Douglas et al., 2004; Yates et al., 2013). Activation of μ-receptors in the nucleus accumbens is especially important for mediating this behavior (Manduca et al., 2016; Trezza et al., 2011). In both rats and non-human primates, pharmacological manipulations with μ-opioid agonists and antagonists increase and decrease play behavior, respec-tively (Beatty and Costello, 1982; Guard et al., 2002; Niesink and Vanree, 1989; Panksepp et al., 1980b; 1985; Trezza and Vanderschuren, 2008; Vanderschuren et al., 1995a; 1995b; 1995c). Studies have shown that social deprivation decreases opioid receptor binding (Schenk et al., 1982) and alter the response to pharmacological treatments affecting the opioid system (Palm and Nylander, 2014a; Smith et al., 2003; 2005; Wongwitdecha and Marsden, 1996).

Central circuitries in the addiction cycle A brief and simplified description of the neurobiology in the addiction cycle is given below, with the aim of putting the brain areas of interest in Papers I-IV into a theoretical framework. The stages of the addiction cycle are cov-ered comprehensively in reviews by Koob and Volkow (2016; 2010)

Different brain areas and neuronal circuitries account for the main effects in the three stages of the addiction cycle. Importantly, the three stages—binge/intoxication, withdrawal/negative effects, and preoccupation/ anticipation—are all interconnected.

In the first stage, when a drug is consumed for its rewarding effects, the mesolimbic dopamine system is activated. The pleasurable and rewarding effect of drug use is the consequence of a sharp increase of accumbal dopa-mine (Di Chiara and Imperato, 1988). Specifically, the shell, and not the core of accumbens, mediates the rewarding effects (Di Chiara et al., 2004). Stimulants, such as amphetamine and cocaine, can act directly on dopamine release whereas other drugs, alcohol for example, indirectly activate dopa-minergic projections by actions on other transmitter networks. These might be, for example, endogenous opioids that act on the opioid receptors of in-hibitory γ-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) and that thereby disinhibit dopamine neurons (Trigo et al., 2010). Repeated binges induce alterations in dopamine dynamics in the dorsal stria-tum, an area involved in habit formation and thereby a shift towards compul-

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sive drug use (Everitt and Robbins, 2016). In addition to these mesolimbic structures, the central nucleus of amygdala also mediates the rewarding ef-fects (Heyser et al., 1999; Hyytia and Koob, 1995; Moller et al., 1997; Robinson et al., 2014). Most drugs activate the hypothalamic-pituitary-adrenal (HPA) axis, which facilitates actions in the mesolimbic reward pathway (Koob, 2008).

In the next stage of the addiction cycle, the repeated drug-binges have de-sensitized the reward system (due to tolerance in, for example, dopamine, serotonin and endorphins). Both drug- and non-drug rewards become attenu-ated (Koob and Volkow, 2016). During withdrawal, the brains stress sys-tem/antireward system, i.e., the HPA axis and the extended amygdala (the central amygdala, bed nucleus stria terminalis, and nucleus accumbens shell) is dysregulated and sensitized (Koob, 2009). The main neurotransmitters during this stage are corticotrophin releasing factor (CRF), dynorphin, and noradrenaline. Their actions, along with the weakened reward system, result in dysphoria, stress, and unease (Koob, 2008). This will motivates the indi-vidual to continue to use the drug to relieve the negative effects of with-drawal, i.e., negative reinforcement (Koob, 2008).

In the last stage, alterations in prefrontal cortical regions are of im-portance. These structures control executive functions such as decision-making and regulation of action, emotions, and impulses (Abernathy et al., 2010); therefore they are highly involved in the decision to take a drug or not. The prefrontal cortex has excitatory (i.e., glutamatergic) neurons that project directly into many areas in the reward networks such as, the VTA, dorsal and ventral striatum and amygdala, and thereby modulates the action of these regions (Koob and Volkow, 2016). During preoccupation and antic-ipation, the glutamatergic system within the prefrontal cortex becomes hy-persensitized. Along with the dysregulation of dopamine and the increase in the brain stress system, this leads to poor self-control (Belin et al., 2009). Consequently, individuals who reach this stage of the addiction cycle have problems resisting impulses and alcohol cravings. After a period of absti-nence, the person becomes preoccupied with using the drug again.

Ethanol Ethanol is a small molecule that is both hydrophilic and lipophilic. Due to its chemical properties, ethanol is widely distributed in the body when ingested and affects almost all organs. Ethanol has no specific target protein. Instead, it acts on many of the reward-related brain areas and associated neurotrans-mitters.

As for almost all other drugs of abuse, ethanol increases dopamine release in the mesolimbic pathway (Boileau et al., 2003; Di Chiara and Imperato, 1985; Weiss et al., 1993; 1996). Since ethanol has such a complex mecha-

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nism of action, it is not fully understood how it increases extracellular do-pamine. One of the proposed mechanisms is that it activates endogenous opioids that bind to μ- and δ-receptors on inhibitory GABA interneurons in the VTA and thereby decreases the inhibiting effects of GABA, which con-sequently facilitates release of dopamine. Furthermore, ethanol can also act directly upon the nucleus accumbens (Trigo et al., 2010).

Ligand-gated ion channels are also involved in the dopamine-releasing actions of ethanol. Ethanol-induced increase of acetylcholine and glycine activates nicotinic acetylcholine receptors in the anterior VTA and glycine receptors in the nucleus accumbens, leading to increased mesolimbic dopa-mine activity (Söderpalm and Ericson, 2013). However, ethanol exerts re-warding effects independent of accumbal dopamine, as evidenced by ani-mals continuing to self-administer ethanol after lesions in dopaminergic neurons in the nucleus accumbens (Fahlke et al., 1994; Lyness and Smith, 1992; Rassnick et al., 1993; Shoemaker et al., 2002).

In this thesis, the focus is on ethanol effects on striatal dopamine and en-dogenous opioid peptides. More detailed information about the mechanism of action of ethanol is covered in reviews by Engel and Jerlhag (2014), Söderpalm and Ericson (2013), Tabakoff and Hoffman (2013).

Effects on dopamine in dorsal and ventral striatum The first indications of a connection between catecholamines and ethanol were in the 1970s when an inhibitor of the catecholamine synthesis sup-pressed ethanol-induced locomotion in rats (Engel et al., 1974), and euphoria and social interactions in humans (Ahlenius et al., 1973). Later, microdialy-sis studies showed that injections of ethanol (Di Chiara and Imperato, 1985) as well as voluntary intake (Weiss et al., 1993), increased accumbal dopa-mine. Within the nucleus accumbens, the shell is responsible for the primary rewarding effects of ethanol, whereas the core is implicated in cue-induced ethanol seeking (Ding et al., 2015; Engleman et al., 2009). In humans, neu-roimaging shows that alcohol increases dopamine in the ventral striatum (Aalto et al., 2015; Boileau et al., 2003) and that the amount released corre-lates to self-reported feelings of intoxication (Ramchandani et al., 2011; Urban et al., 2010; Yoder et al., 2007). Furthermore, alcohol downregulates striatal dopamine D2 receptors, and alcohol craving in alcoholics is associat-ed with a low availability of D2 receptors in the ventral striatum (Heinz et al., 2004; Hietala et al., 1994; Volkow et al., 1996).

The early studies regarding acute ethanol exposure and dopaminergic ac-tivity in the dorsal striatum were inconsistent. Di Chiara and Imperato (1985) showed that moderate to high doses increase dopamine but a low dose has no significant effect. Blanchard et al. (1993) presented conflicting results with increased dopamine at low doses. The inconsistency in results could be due to interregional differences within the dorsal striatum, as the

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ethanol-induced increase in extracellular dopamine occurs in the dorsomedi-al, and not the dorsolateral part (Vena et al., 2016). Dopaminergic activity in the dorsolateral striatum seems to be more involved in the shift to habitual alcohol seeking (Corbit et al., 2012) and influence the motivation to obtain alcohol when it requires high levels of effort (Spoelder et al., 2017). Thus, the dorsolateral part is involved when the consumption behavior becomes increasingly compulsive. In human studies, the ventral striatum in social drinkers is activated when presented with an alcohol cue, whereas the dorsal striatum becomes active in heavy drinkers (Vollstädt-Klein et al., 2010). Furthermore, alcohol-dependent patients have an imbalance between goal-directed and habitual control with a decreased activity in the ventromedial prefrontal cortex and anterior putamen (i.e., regions implicated in goal-directed learning), and increased activity in the posterior putamen (i.e., a region involved in habit learning) (Sjoerds et al., 2013).

Alcohol consumption is indeed modulated by pharmacological agents af-fecting dopamine transmission (Gilpin and Koob, 2008). However, the ad-verse effects of dopamine antagonists (e.g., anhedonia and extrapyramidal actions) have made clinical use difficult. Furthermore, dopamine agonists do not induce relief of the dysphoria caused by the low dopamine activity after excessive drinking (Engel and Jerlhag, 2014; Kosten et al., 2002). There is attempt to use more dynamic dopamine stabilizers, such as aripripazole and (-)-OSU6162, in the treatment of alcohol use disorders (Ingman et al., 2006; Steensland et al., 2012).

Effects on endogenous opioids On the Swedish market, two of the four drugs available for alcohol use dis-order targets opioid receptors. The first of these, naltrexone, is an unspecific antagonist with the highest affinity to μ-receptors and slightly higher affinity to κ- over δ-receptors (Nutt, 2014). The other drug, nalmefene, modulates the opioid system, with antagonistic properties to μ- and δ-receptors and partial agonism to κ-receptors (Bart et al., 2005). Nalmefene exhibits greater affinity to κ- over δ-receptors compared to naltrexone (Emmerson et al., 1994; Michel et al., 1985).

Opioid receptors are located in all brain areas of importance for reward mechanisms (Le Merrer et al., 2009). As mentioned in “Ethanol”, opioids modulate reward and reinforcement. The μ- and δ-receptors account for the positive effects of the ethanol intake and the κ-receptors act counterbalanc-ing (Nutt, 2014). The agonists for μ- and δ-receptors mediate dopamine re-lease in nucleus accumbens, induce place preference and are self-administered (Devine et al., 1993; Devine and Wise, 1994; Spanagel et al., 1990). The κ-receptor agonists decrease accumbal dopamine release, pro-duce aversive effects in preference tests, and are not self-administered

22

(Mucha and Herz, 1985; Mulder et al., 1984; Spanagel et al., 1990; Werling et al., 1988).

The literature describing ethanol-induced effects on opioids is not conclu-sive. However, in rodents, ethanol releases β-endorphin from the hypothala-mus, pituitary, nucleus accumbens and VTA, and increases enkephalins in the nucleus accumbens (de Waele and Gianoulakis, 1993; Gianoulakis, 1990; Jarjour et al., 2009; Lam and Gianoulakis, 2011; Olive et al., 2001). In humans, ethanol intake elevates β-endorphins in the nucleus accumbens and orbitofrontal cortex of both healthy controls and heavy drinkers (Mitchell et al., 2012). Dynorphin is also increased in the nucleus accumbens and central amygdala after acute ethanol exposure (Lam et al., 2008; Marinelli et al., 2006). In a non-dependent state, the κ-system suppresses ethanol reinforce-ment. However, after chronic ethanol use, the dynorphin/κ-system becomes upregulated and during withdrawal, it is involved in the dysphoria that leads to the negatively reinforcing effects of ethanol (Koob, 2008; Sirohi et al., 2012).

N/OFQ counteracts the CRF-induced stress response in the brain (Ciccocioppo et al., 2014; Cruz et al., 2012) and it has been hypothesized that NOP agonists could potentially attenuate the negative effects during withdrawal (Witkin et al., 2014). Preclinical studies have supported the hy-pothesis, with agonists able to reduce intake, prevent somatic effects of withdrawal, and block cue- and stress-induced alcohol reinstatement (Aziz et al., 2016; Ciccocioppo et al., 2004; 1999; Economidou et al., 2011; Martin-Fardon et al., 2000; 2010). However, a recent neuroimaging study found no differences in NOP receptor binding for alcohol-dependent individuals and healthy controls (Narendran et al., 2017).

Amphetamine Amphetamine exerts multiple actions on the dopamine synapse that leading to increased levels of extracellular dopamine (Figure 3). Amphetamine binds to DAT in the cell membrane and competes with dopamine binding (Sulzer, 2011). Furthermore, amphetamine can be transported into the presynapse where it disrupts vesicular storage of dopamine through blockade of VMAT and/or collapse of the pH gradient in the vesicular membrane, leading to vesicular leakage (Sulzer, 2011). Together, these events lead to reversed DAT transport and increased levels of dopamine in the synaptic cleft. Other mechanisms of action have also been proposed such as inhibition of MAO and increased dopamine synthesis (Sulzer, 2011).

23

Figure 3. Mechanism of action of amphetamine. Amphetamine increases the synap-tic concentration of dopamine by; reversing DAT; disrupting the storage of dopa-mine in the synaptic vesicles; and increasing the synthesis of new dopamine (Sulzer, 2011). Abbreviations; D, dopamine receptor; DAT, dopamine transporter; DOPA, dihydroxyphenylalanine; DOPAC, dihydroxyphenylacetic acid; VMAT, vesicular monoamine transporter. Modified with permission from Stina Lundberg, Dept. of Pharmaceutical Biosciences, Uppsala University.

DOPA decarboxylase

Tyrosine hydroxylase

DOPA

Tyrosine

MAOA

D2

Gαi DAT

VMAT

DAT DAT

DOPAC

-+

-+

+

Aldehyde dehydrogenase+

DD D

Dopamine Amphetamine

24

Methodology

Maternal separation – simulation of early life stress The environment of a rat pup is almost exclusively determined by the inter-action with its mother, as the pup and its littermates, live in the nest until weaning. The most common strategy to simulate an adverse environment, and induce early life stress, is to disturb the maternal care. This is usually done by separating the mother from the pup (Figure 4A). The separation can be litter-wise or individual, varying in duration from 180-360 minutes; long-er separations up to 24 hours are usually called maternal deprivation (Knop et al., 2017). The frequency and the timing of the separation can be modulat-ed. The separation can be either daily or intermittent and the timing can be during the first two weeks or until weaning (Molet et al., 2014). Another important aspect for this experimental model is the choice of control group. Litters reared under conventional animal facility standards or left non-handled (i.e., undisturbed) can control for the repeated handling and separa-tions, but it is not possible to distinguish between handling and separation effects (Nylander and Roman, 2013). To only investigate the effect of the separation, one could use groups exposed to either brief handling (i.e., 1-5 minutes of separation) or short maternal separation (i.e., 15 minutes) (Nylander and Roman, 2013). Furthermore, short separations between dam and pups provide a more naturalistic environment; in the wild, the dam leaves the nest regularly for shorter periods (Grota and Ader, 1969). Mater-nal separation models have been criticized for their lack of reproducible results, but the outcome of the experiment depends on all the above-mentioned factors, i.e., type, duration and frequency of separation as well as timing and the choice of control group. Therefore, maternal separation is not a single model of early life stress, but instead several models of separation that fall under the same umbrella.

Another way to induce early life stress, without separation, is to limit the nesting and bedding material in the cage; this stresses the dam which results in fragmented and unpredictable maternal care (Molet et al., 2014). Natural variance in the quality of maternal care is a factor difficult to control for, but important since it per se affects the offspring (Curley and Champagne, 2016). However, this factor is independent of early life stress model and can only be controlled for by scoring the dams’ maternal behavior.

25

Figure 4. Manipulations of housing conditions (Papers I-II). A) Early life stress was assessed in Paper I by 360 minutes of maternal separation. 15 minutes of maternal separation was used to control for handling effects, and animal-facility reared (i.e., left undisturbed) rats were used as a conventional control. B) Effects of housing conditions during adolescence were assessed in either group-housed or single-housed rats in Paper II.

Experimental housing conditions – simulation of social environments The laboratory rat (Rattus norvegicus) is a social species, that in the wild, lives in large colonies with defined hierarchies; if laboratory rats are placed in a semi-naturalistic environment they readily revert to their wildlife behav-ior (Berdoy, 2003). The knowledge of how sensitive rats are to environmen-tal conditions has been used in biomedical research to develop models that alter the social environment (i.e., housing conditions) (Figure 4B). By simp-ly manipulating the number of animals in the same cage, it is possible to induce stress by crowding or isolation (Miczek et al., 2008). The hierarchical system can also be manipulated to induce social stressors, such as social instability, defeat, and subordination (Miczek et al., 2008). Enrichment of the housing conditions can also be used to study the effects of a secure and stimulating environment (Simpson and Kelly, 2011). Even when the aim is not to study the consequences of the environmental setting, it is still im-

A

BAnimal facility rearing

15 minutes ofmaternal separation

360 minutes ofmaternal separation

Group housing

Single housing

26

portant to control the housing conditions throughout the entire experiment to avoid confounders. Uncontrolled for, inconsistencies in the animals’ social conditions can potentially affect the outcome of an experiment, making it difficult to reproduce results between different laboratories.

Alcohol exposure – simulation of adolescent binge-like drinking Several aspects need to be considered when exposing adolescent rats to eth-anol, depending on research question. To investigate drinking phenotypes in a voluntary intake model with high face validity (i.e., to mimic aspects of human intake of alcohol), then operant self-administration or home-cage drinking (non-operant self-administration) are the preferred methods. Etha-nol-naïve rats do not readily self-administer ethanol with the operant tech-nique and therefore extensive training or pre-exposure is necessary before the actual experiments can begin (June and Gilpin, 2010). Adolescence is a fairly short period in a rat’s life and operant self-administration is a time-consuming method; therefore, home-cage drinking is more frequently used because rats readily consume ethanol via this route of administration (Sanchis-Segura and Spanagel, 2006). However, to assess the individual intake of ethanol, this model usually involves single housing which induces stress and long-lasting neurobiological consequences in adolescent rats (Siviy and Panksepp, 2011).

Models that involve forced exposure have weaker face validity but facili-tate the study of neurobiological outcomes of ethanol exposure in a con-trolled way. Both intragastric administration (Figure 5A) and intraperitoneal injections of ethanol activate the HPA-axis (Ogilvie et al., 1997) and are therefore possibly stressful events. However, Turner et al. (2012) have shown that a daily gavage dose not negatively affect animals welfare and Hoffman (2014) showed that gavage had less impact than intraperitoneal injections on corticosterone levels, as a measure of stress. Another method to minimize stress-related handling is the use of intragastric cannulas (Ogilvie et al., 1997). However, as mentioned above, adolescence in rats only lasts a few weeks and the recovery after such an operation has a large impact on both the time and length of the ethanol exposure. Another model that can be used is ethanol exposure through vapor chambers, which requires neither single housing nor handling, but inhalation does not mimic human ingestion of alcohol.

27

Voluntary alcohol drinking – evaluation of consumption behavior In this section, only home-cage drinking (Figure 5B) will be described; op-erant self-administration (Figure 5C) will be explained in “Operant self ad-ministration – evaluation of drug taking behavior”.

Figure 5. Drug exposure paradigms (Papers I-IV). A) Orogastric exposure i.e., ga-vage (Papers III-IV), B) Voluntary ethanol drinking in a two-bottle, free-choice paradigm (Paper I), C) Intravenous self-administration in operant boxes (Paper IV).

Home-cage drinking is a convenient way of evaluating consumption be-havior as it can be easily implemented by adding bottles with ethanol into the cage (Richter and Campbell, 1940). It has both the face and construct validity of human alcohol consumption and pharmacological treatments can prevent excessive drinking, indicating a predictive validity (Sanchis-Segura and Spanagel, 2006). However, to induce addiction in outbred rats by volun-tary drinking is challenging and therefore, home cage drinking is most suita-ble for assessing acquisition and habitual drinking in these rats.

A B

Operantself-administration

Voluntary drinkingOrogastric

exposure

C

28

In most alcohol intake models, the animal has a free choice of drinking ethanol with water and intake of food ad libitum so that the alcohol is con-sumed for its pharmacological effects and not its caloric value or thirst (Bell et al., 2017). Sweetened solutions are sometimes added to increase the amount of liquid ingested, but that makes it difficult to distinguish between the preference for sweet taste and the psychopharmacologic actions of the ethanol (Sanchis-Segura and Spanagel, 2006). In low concentrations (< 4% v/v), ethanol by itself can have a sweet taste (Sanchis-Segura and Spanagel, 2006), and hence it is important to use pharmacological relevant concentra-tions, i.e., >10%. Rodents are nocturnal animals with their highest level of eating, drinking and general activity in the dark phase of their circadian cy-cle. Thus, ethanol consumption increases when administered in the dark (Crabbe et al., 2011; Rhodes et al., 2005).

The access to alcohol can be continuous or intermittent during the drink-ing period; restricting the availability to every other day increases alcohol intake and preference (Wise, 1973). In the intermittent paradigms, the access can be either unlimited or limited. Unlimited access makes it difficult to determent if the intake has led to relevant blood alcohol concentrations or if the animal sporadically sips from the ethanol bottle and hence, not reaching significant pharmacological effects (Becker, 2013). Limited access is also better when analyzing tissues, since it decreases the inter-individual differ-ences. This leads to better relation between the results (e.g., tissue levels of peptides) and the amount of ethanol intake.

Operant self-administration – evaluation of drug-taking behavior Operant self-administration (Figure 5C) is frequently used for studying vol-untary intake of psychostimulants (e.g., cocaine and amphetamine) since rats easily learn the operant technique with this type of substances. The disad-vantage with operant self-administration is that it is not as convenient as home-cage voluntary intake. It requires both training and more sophisticated equipment, i.e., operant self-administration boxes equipped for intravenous drug delivery. Self-administration boxes are also known as Skinner-boxes, named after their inventor, B.F. Skinner. He developed these boxes for oper-ant conditioning where the animal learns that a certain behavior (e.g., a lever press) is associated with a stimuli (e.g., a light signal) and positive rein-forcement (e.g., drug infusion) (Skinner, 1938). Operant self-administration facilitates observations of many behaviors, for example consumption behav-ior, reward efficacy, extinction and cue-induced reinforcement. In this thesis, consumption behavior and reward efficacy were studied.

Consumption behavior is mainly studied through fixed ratio (FR) sched-ules. In this type of trial the drug is delivered after a preset number of re-sponses (e.g., a number of lever presses). To examine oral ethanol intake with operant boxes, low FR schedules (i.e., one press on the active lever,

29

FR1) are required to maintain self-administration. In contrast, higher FR schedules (e.g., FR3) are used for drug delivery systems with an immediate drug effect (e.g., intravenous amphetamine infusions) (Sanchis-Segura and Spanagel, 2006).

Progressive ratio (PR) trials are used to investigate reward efficacy. This method was first developed by William Hodos (1961) when he tested the reinforcing properties of sweetened milk in rats. In PR trials the response requirements increase systematically for each reward and therefore give an estimation of the maximum response for maintenance of self-administration (Richardson and Roberts, 1996). The point in the escalating series of re-sponse requirements at which the animal stop responding is called the break-point. This value reflects the maximum effort the animal is willing to make in order to receive a reward (e.g., a drug infusion) (Richardson and Roberts, 1996). Different reinforcers produce different breakpoint values. For exam-ple, psychostimulants have high breakpoints values (Richardson and Roberts, 1996) whereas alcohol has a lower one (June and Gilpin, 2010).

In vivo high-speed chronoamperometry – analysis of dopamine A variety of methods (e.g., electrochemical recordings, microdialysis, and neuroimaging) can be used to measure in vivo dopamine. In this thesis, do-pamine was measured with high-speed chronoamperometry (Figure 6), an electrochemical technique. A small electrode is placed into the brain area of interest and a potential applied to the electrode causing any molecules in contact with the electrode to be oxidized (Michael and Wightman, 1999). Not all molecules in the extracellular fluid can be oxidized, therefore the application of this method depends on the analyte of interest (Gerhardt and Burmeister, 2000). However, with the proper potential, dopamine can be oxidized to form an o-quionone that can be reduced back to dopamine. This reaction results in measurable elicitations of Faradaic currents that are pro-portional to the analyte concentration (Gerhardt and Burmeister, 2000). The ratio between the oxidation and reduction currents creates an electrochemical fingerprint of the analyte (Gerhardt and Burmeister, 2000). The way of ap-plying the potential over the electrode differs depending on the electrochem-ical technique. The simplest way is to use a constant and fixed potential (re-ferred to as amperometry in the literature), which leads to continuous meas-urements of currents at the electrode and a good temporal resolution (Michael and Wightman, 1999). However, the chemical selectivity is poor. In high-speed chronoamperometry the voltage is applied to the electrode in a square-wave manner that gives the opportunity for oxidation and reduction of the molecule, and hence the possibility to identify the analyte (Michael and Wightman, 1999). Nevertheless, it should be mentioned that dopamine and other biogenic amines (e.g. noradrenaline, serotonin) could be difficult to distinguish from each other with this method. However, the chronoam-

30

perometric recordings in this thesis are performed in the dorsal striatum where dopamine is the mostly abundant and dominant biogenic amine. Mi-crodialysis, a third technique, allows collection and analysis of samples con-taining a number of different molecules in both basal state and after a phar-macological or physiological challenge (Gerhardt and Burmeister, 2000). The main advantage with high-speed chronoamperometry over microdialysis is its spatial and temporal resolution (Gerhardt and Burmeister, 2000).

Figure 6. Overview of high-speed chronoamperometry (Paper IV). A) The micro- and reference electrodes are inserted into the dorsal part of striatum through a stereo-taxic operation in anesthetized rats. B) K+ is released (I) into the brain area of inter-ested and causes a neuronal depolarization (II), and release of dopamine (III). A square-wave potential is applied to an electrode, which then causes dopamine to oxidize (IV). The current generated is proportional to the concentration of released dopamine. C) A schematic example of a trace of oxidation current from a rat receiv-ing amphetamine. D) A close-up of a peak showing how amplitude and T80 values are calculated. Figures B) and C) is modified with permission from Stina Lundberg, Dept. of Pharmaceutical Biosciences, Uppsala University (B) and Sara Palm (2014) (C).

The disadvantage is that measurements of resting or basal levels of dopa-mine are difficult to perform due to low extracellular levels (Gerhardt and Burmeister, 2000). To overcome this limitation, measurements of neuronal

A

Time (s)

[μM

]

Amplitude

T80

Time (s)

BNH2OH

OH

Dopamine

K+

K+ K+K+

K+

K+

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II.

III.

IV.

+2H+

2e-2e-

O

NH2O

C D

Amphetamine

References

[μM

]

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release can be done through chemical (e.g., potassium-induced) or electrical stimulation of the neurons (Michael and Wightman, 1999) in pharmacologi-cally untreated or treated subjects. Commercial systems for high-speed chronoamperometry can easily measure and analyze the neuronal release of for example dopamine. This is displayed as a peak where the peak amplitude relates to the amount of released dopamine, and the time for the signal to decay 80% (T80), is a measurement of dopamine re-uptake (Zahniser et al., 1998) (Figure 3D).

Real-time polymerase chain reaction – analysis of gene expression The major breakthrough in gene research occurred in the 1950´s when Rosalind Franklin produced an X-ray diffraction picture of deoxyribonucleic acid, DNA (Franklin and Gosling, 1953). With the help of her data, Watson and Crick solved the double helix structure of DNA (Watson and Crick, 1953). In 1962, Watson and Crick shared the Nobel Prize with Maurice Wil-kins for this discovery (Nobelprize.org). Another groundbreaking, and Nobel Prize awarded (Nobelprize.org), discovery was that of polymerase chain reaction (PCR) (Mullis and Faloona, 1987). PCR is a process whereby spe-cific segments of DNA (e.g., genes) can be amplified into multiple copies. Briefly, the process involves several steps of increasing and decreasing tem-perature in a solution containing; DNA, nucleotides, gene-specific primers, and thermostable DNA polymerase. The reaction begins by separating the double helix of the DNA to allow the attachment of primers designed for specific parts of DNA. Thereafter, the DNA polymerase begins to synthesize new DNA strands with the primers as starting points. After synthesis, the original DNA-molecule has now been duplicated and in forthcoming cycles, the designated DNA is exponentially amplified.

In 1993, the first real-time PCR was made (Higuchi et al., 1993). Before real-time PCR instruments were available, the quantification of DNA had to be done after the PCR; with real-time PCR, the amplification of DNA occurs simultaneously and combines both amplification and detection (Wong and Medrano, 2005). Real-time PCR is also known as quantitative PCR (qPCR). Real-time measurement is made possible by incorporation of fluorescent probes (e.g., SYBR green) that bind to the copied DNA; as the number of PCR cycles increases the fluorescent signal from amplified DNA intensifies (Figure 7). Real-time PCR can be divided into four phases; a linear ground phase in which the fluorescence is below background noise; an exponential phase with doubling and accurate measurement of the PCR product at every cycle; a linear phase during which the PCR begins to slow down and gives high variability between runs; and finally, a plateau phase, in which the reac-tants are consumed and no more products can be synthesized. The strength

32

of the fluorescence during the exponential phase is turned into quantifiable numerical values (Ishmael and Stellato, 2008). Genomic DNA amplification can provide important information, but does not reveal if the selected gene is expressed in a specific tissue.

Figure 7. Reverse transcriptase real-time PCR (Paper I). A) Reverse transcriptase PCR. A RNA molecule (1) is used for the first-strand cDNA synthesis (2) that leads to a cDNA:RNA hybrid (3). The hybrid will thereafter act as the first template in the forthcoming amplification of cDNA (4). B) SYBR Green incorporates to double-stranded DNA and emits fluorescent signals, (1), when the DNA breaks during de-naturation SYBR Green is released and is no longer able to emit (2). In the extension phase, double-stranded DNA is yet again produced and fluorescence re-appears (3). When the PCR cycle is finished, the fluorescent signal is twice as intense as before (4). C) The four phases—ground, exponential, linear and plateau—in a real-time PCR run.

To find out how gene expression is affected by different conditions, it is necessary to convert and amplify messenger RNA (mRNA) to complemen-tary DNA (cDNA) with reverse transcriptase before amplification by PCR (Bank et al., 1972; Ishmael and Stellato, 2008; Temin and Mizutani, 1970; Verma et al., 1972). This type of qPCR is then called reverse transcriptase-PCR (RT-qPCR).

A disadvantage with qPCR its lack of spatial resolution, which is im-portant in the analysis of complex tissues such as brain samples. To over-come this obstacle, one could use in situ hybridization; however, quantitative measurements with this method are not as accurate as those with qPCR.

The most recent invention in PCR methodology is absolute quantitative digital PCR (dPCR), which is based on the principle that a sample is diluted into a very large number of reaction chambers in which the PCR reaction can occur. In the beginning of the reaction there should be at most only one mol-ecule in each chamber, and by counting the chamber in which a PCR reac-tion has occurred the absolute number of DNA can be calculated (Baker,

1.

2.

3.

4.

Reverse transcriptase

PCR

RTase

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B

Nucleotides

Denaturation

Extension

Polymerization

1.

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4.

Flou

resc

ence

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ThresholdGround

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ar

}

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2012). Besides the benefit of absolute quantification, it does not need any baseline estimation of the fluorescence, a factor that sometimes leads to am-plification bias in qPCR (Cao et al., 2017). However, dPCR is in its early days, hence, it is much more expensive than qPCR and it is not yet widely used.

Radioimmunoassay – quantification of peptides Various immunoassays can be used to measure the concentration of macro-molecules in tissues and body fluids, and in this thesis, radioimmunoassays were used to measure opioid peptides and corticosterone (Figure 8). Two pioneers of radioimmunoassays were Solomon Berson and Rosalyn Yalow. During the 1950s they developed an assay that could detect insulin levels in human plasma (Yalow and Berson, 1959). For this accomplishment, Yalow was awarded the Nobel prize in 1977 (Nobelprize.org). The method has been developed further and today, proteins and peptides from a variety of tissues can be measured with radioimmunoassay.

Figure 8. Peptide analysis (Papers II-III). A) Frozen whole brains are stabilized to avoid enzymatic degradation (Paper III). B) Peptides are extracted from brain tissues by ion-exchange chromatography; samples are placed in columns filled with Se-phadex gel and the peptides are eluted by buffers with varying ion strength. C) The purified peptides are mixed with radioactive (125I) labeled peptides and a primary antibody. The immunoreactive level of antibody-bound peptide is thereafter meas-ured in a γ-counter.

The general principle of the method is that antigens labeled with radioiso-topes (e.g., 125I) bind to specific antibodies and form a labeled antigen-antibody complex. Unlabeled antigen from the tissue of interest competes with the labeled antigen and consequently inhibits the binding of the labeled antigen (Berson and Yalow, 1968). By using solutions of known concentra-tions of unlabeled peptides, a standard curve can be obtained and used to calculate unknown concentrations in a tissue sample.

125I labeled peptideUnlabeled peptide

Primary antibody

Bound peptide

Sephadex gelSample

Extractedpeptides

A

Buffers

B C

34

The advantages with radioimmunoassays are the high specificity and sen-sitivity, the possibility to measure different opioid peptides in several brain areas in the same animal, and the ease of analyzing a large number of sam-ples in a time efficient way. The disadvantage with the method is the risk of cross reactivity with similar peptide fragments.

There are some other, general problems in analyzing tissue levels of pep-tides. When dissecting tissues, it is difficult to separate specific subregions within brain areas (e.g., the basolateral or the central part of amygdala), therefore the analysis is usually conducted on the whole region. Post-mortem degradation of peptides is another problem with dissected tissues and careful handling of the samples during the purification of the peptides is crucial. However, the post-mortem degradation can be reduced by stabilizing the tissue by conductive heat transfer using the Stabilizor T1 instrument (Dena-tor AB) (see Tissue stabilization) (Segerström et al., 2016).

Opioid peptides in the brain can also be analyzed with in vivo microdialy-sis, which allows for real time measurements of the basal levels and the ef-fect of pharmacological and physiological challenges. The disadvantage with this method is that usually a single brain structure is measured in the same animal and the method is much more time consuming than radioimmunoas-says.

35

Aims

The overall aim was to investigate the effect of stressful environments or alcohol exposure early in life on the central nervous system and/or drug tak-ing later in life.

The specific aims were to examine the effects of: • Early life stress on opioid gene expression and subsequent voluntary

ethanol intake in adult rats. • Housing conditions on endogenous opioid peptides in the brain and the

pituitary of adolescent rats. • Episodic binge-like ethanol exposure during adolescence on the endoge-

nous opioid peptides in the brain and the pituitary. • Ethanol exposure in adolescence on in vivo dopamine dynamics in dorsal

striatum and amphetamine self-administration behavior in adulthood

36

Methods

An overview of the experiments is presented in Figure 9.

Figure 9. Experimental outline for Papers I-IV. A more detailed outline is presented in the respective Papers (I-IV). Words in bold represent the measured outcome pre-sented in this thesis. Early life refers to the period before weaning, postnatal days 0-21. Adolescence represents the period between postnatal days 22-63.

Animals Pregnant Wistar (Harlan Laboratories B.V., the Netherlands (Papers I, III and IV)) and Sprague Dawley (Scanbur BK AB, Sweden (Paper II)) rats arrived at gestational day 15 (Papers I, III and IV) or 17 (Paper II). The dams were kept on a 12 hours light/dark cycle in type IV cages (59 × 38 × 20 cm) containing wood chip bedding and nesting materials. Animals born on the

Paper I

Paper II

Timeline

Paper III

Maternal separation Voluntary ethanol drinkingOpioidgenes

Opioid peptides

Episodic ethanol binge exposureOpioid

peptides

Episodic ethanol binge exposure

Dopaminerecordings

DopaminerecordingsAmphetamine

self-administrationSucrose

self-administration

Paper IV

Early life Adolescence Adulthood

Experimentalhousing conditions

Opioidpeptides

37

same day (postnatal day 0) were sexed and cross-fostered to avoid biological littermates, each litter contained the same number of males and females. In Paper I, the litters were either reared under standard animal facility condi-tions or exposed to maternal separation (see Maternal separation). In Papers II-IV, male rats were randomly assigned to the different experimental groups immediately after weaning and group-housed on a reversed light/dark cycle before the experiments. All animals were group-housed during adolescence unless otherwise stated. In adulthood, the animals were group-housed with the exception of Paper I where single housing was necessary due to the ex-perimental procedure.

All animals had access to water and food ad libitum unless otherwise stat-ed. Animal weights were regularly monitored throughout the experiments. The cages were kept in temperature- (22 ± 1°C) and humidity- (50 ± 10%) controlled cabinets and the animal rooms had a masking background noise to minimize unexpected sounds.

All experimental procedures and animal care followed the guidelines of the Swedish Legislation on Animal Experimentation (Animal Welfare Act SFS1998:56) and the European Communities Council Directive (86/609/EEC) and were approved by the Uppsala Animal Ethical Commit-tee.

Maternal separation (Paper I) Litters were randomly assigned to one of the three rearing environments: 15 or 360 minutes of maternal separation (MS15/360) or conventional animal facility rearing (AFR). The separations of litters and dam were performed during the light period. Firstly, the dam was removed from the home cage to a type II (26 × 20 × 14 cm) macrolon cage containing wood chip bedding. Secondly, the litter was moved from the home cage to a type II cage (with bedding) and transferred to a heating cabinet (30.4 ± 0.2°C, 52.1 ± 0.7 hu-midity) in an adjacent room. The dams of the MS15 groups remained in the type II cage during the 15 minutes of separation and the pups returned to the home cage before the return of the dam. The dams in the MS360 group were returned to the home cage during the 360 minutes of separation. The separa-tions were performed every day from postnatal day 0 to 21. The animals in the AFR group were left undisturbed with the exception of care taking and weighing of the litter. On postnatal day 22, all animals were weighed and weaned. The separations and care taking was always performed in the same animal rooms and by the same person

38

Adolescent housing conditions (Paper II) At postnatal day 22, the rats were randomly assigned to three experimental groups: (i) a longer period (seven days) of single housing; (ii) group-housing with 30 minutes of short single housing before decapitation; and (iii) group housing until decapitation (i.e., the controls).

Adolescent ethanol exposure (Paper III-IV) Between four and nine weeks of age, animals received orogastric (i.e., ga-vage) administration of either water or 2 g/kg (20% v/v) ethanol diluted with water. Administrations were given at 09:00 on three consecutive days fol-lowed by four days without treatment. Animals were kept group-housed during the entire ethanol exposure period.

Adult voluntary ethanol intake (Paper I) The rats were individually housed in cages containing wood chip bedding and a wooden house. For three consecutive days per week, the rats had free access to both ethanol and water. During the first week, the rats were given 24 hours ad libitum access to 5% (v/v) ethanol and two hours during the second week. Thereafter ethanol concentration was increased to 20% (v/v) and access was limited to two hours. Ethanol and water were changed every day during the three days and the bottle position was altered daily to avoid position preference. At the end of each drinking session, ethanol and water intake was measured by weighing the bottles.

Operant self-administration (Paper IV) Operant boxes apparatus Self-administration was conducted in sound-attenuated operant boxes (MED Associates Inc., VT, USA) equipped with two stimulus lights above two retractable, stainless steel levers. A white house-light placed on the wall opposite the levers was on during the entire session; a ventilating fan operat-ed throughout the sessions and served as a masking noise. Intravenous solu-tions were delivered by infusion pump; a plastic syringe placed in the pump was connected to the implanted catheter through CoEx tubing (Harvard Ap-paratus, Kent, UK) and protected by a flexible metal leash. Experiments were run and data collected with the MED-PC software. The experiments were conducted in the same operant boxes for both sucrose training and am-

39

phetamine sessions. Ethanol-treated animals and water controls were pro-cessed simultaneously throughout all phases of the self-administration pro-cedure.

Sucrose training Food restriction was initiated following the last session of ethanol exposure and was maintained throughout the sucrose-training period. Animal weights were carefully monitored and were not allowed to decrease more than 15 % after the commencement of food restriction. After 48 hours of food re-striction, the training schedule to self-administer 45 mg sucrose food pellets (5-TUL, TestDiet, MO, USA) on a fixed ratio-1 (FR1) was initiated. The criteria for fulfilled self-administration training were 100 active lever presses within 30 minutes and a specificity >0.85 for the active lever.

Intravenous amphetamine self-administration Intravenous catheters (CamCaths, Ely, UK) were implanted into the right jugular vein under isoflurane (Forene Abbott, Sweden) anaesthesia. Postop-erative analgesia (buprenorphine and carpofen) and antibiotic (amoxicillin) were administered. The catheters were flushed with a heparin solution before and after each session, and a heparinized glycerol lock solution was used over weekends. Catheter patency was tested before the start and at the end of the study with an infusion of the short-acting anaesthetic agent propofol.

After a minimum of four days of recovery from surgery, rats from both the ethanol and water groups were allowed to self-administer amphetamine (d-amphetamine sulfate, Sigma-Aldrich, MO, USA) on a daily 60 minutes FR3 schedule of reinforcement. Three responses on the active lever resulted in an infusion of amphetamine (0.1 mg/kg/infusion) and a 10 seconds time-out period was initiated during which both levers were retracted and a white stimulus light above the active lever was turned on. The lever designated as the active one was switched between sucrose training and intravenous self-administration. The maximum number of rewards during the 60 minutes baseline sessions was set to 20. A press on the inactive lever had no pro-grammed consequences, but the activity was recorded by the software for statistical analysis.

After five 60 minutes FR3 amphetamine (0.1 mg/kg/infusion) baseline sessions to familiarize the animals with the amphetamine effects, operant requirements were switched to the PR format. Under this schedule of rein-forcement, the response requirement started at one press on the active lever and escalated for each drug infusion delivered according to following scheme: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, 737, 901 see Richardson and Roberts (1996). The PR schedule was tested at two doses (0.1 and 0.05 mg/kg per infusion)

40

for two consecutive days each. The sessions ended when one hour had passed since the last reward or after a maximum session time of four hours. The breakpoint was defined as the total number of infusions during the ses-sion. Additionally, to test the dose-response function on an FR schedule the unit dose of amphetamine (0, 0.025, 0.05 or 0.1 mg/kg/infusion) was varied and each dose was tested for three consecutive 90 minutes FR3 sessions. The first session of each dose in PR and dose-response trials was considered an acclimatization one and these data collected were not used in the statistical analysis.

High speed chronoamperometry (Paper IV) Dopamine recordings were conducted using carbon fibre microelectrodes (Quanteon, KY, USA). A high-speed chronoamperometric protocol was used (550mV, 1 Hz sampling rate, 200 ms total) via a FAST16-mkII recording system (Fast Analytical Sensing Technology, KY, USA). Electrode-pipette assemblies were prepared and calibrated immediately before in vivo record-ings, as previously described (Gerhardt and Hoffman, 2001; Littrell et al., 2012). Briefly, electrodes were coated with Nafion and calibration were conducted with cumulative additions of ascorbic acid and dopamine. Elec-trodes had a detection limit of 0.0237±0.0037 μM and a selectivity of 3864.35±881.16 for dopamine over ascorbic acid. Responses to dopamine were linear, with an average correlation coefficient (R2) of 0.827±0.025. The average reduction/oxidation ratio was 0.628±0.011, which indicates specific dopamine detection (Gerhardt and Hoffman, 2001). After calibration, a mi-cropipette filled with isotonic potassium chloride solution was affixed with the tip 150-200 μm from the recording site of the electrode.

Animals were anaesthetized with Inactin and body temperature main-tained with a thermostatic heating pad. The electrode-pipette assembly was stereotaxically placed in the dorsolateral striatum (Anterior-Posterior: +1.0, Medial-Lateral: +3.0, Dorsal-Ventral: -4.2 mm) and an Ag/AgCl reference electrode placed in the brain contralaterally and remotely from the electrode-recording site.

After one hour post-surgical stabilization of the electrode and surrounding tissue, 100 nl of potassium chloride solution was locally ejected using pres-sure ejection (PicoSpritzer III, Parker Hannifin Corporation, NJ, USA, ejec-tion pressure <22 psi, ejection time < two seconds) and the subsequent do-pamine release was detected by the electrode as a peak of rising dopamine concentration. Ejections were repeated every 10 minutes until three succes-sive consistent dopamine releases were recorded for use as baseline refer-ence peaks. Five minutes after the third reference peak was evoked, a single 2.0 mg/kg dose of amphetamine was injected via the tail vein. Five minutes post-injection of amphetamine, dopamine release was evoked again and this

41

was then repeated every 10 minutes until 55 minutes post-injection. After the measurements, the animals were killed and the brains removed and frozen for subsequent histological identification of electrode location.

Quantitative polymerase chain reaction (Paper I) To purify and isolate RNA from brain tissues, All Prep DNA/RNA /miRNA Universal Kit (Qiagen, Sweden) was used according to the manufacturer’s instructions. Isolated RNA was quantified using a Nanodrop ND 1000 spec-trophotometer. RNA was converted to cDNA by using the QuantiTect Re-verse Transcription Kit (Qiagen AB, Sweden) including a genomic wipeout reaction. The synthesis of cDNA was performed at 42°C for 30 minutes and inactivated at 95°C. Newly synthetized cDNA was diluted and stored at -20°C. The diluted cDNA was used for qPCR (CFX96, Bio-Rad, Sweden) to assess the expression of the genes of interest – Pomc, Penk, Pdyn, Oprm1, Oprd1 and Oprk1 – as well as the housekeeping genes Actb, Gapdh and RpI19. Primers were designed using Primer 3 (http://frodo.wi.me.edu/) and crosschecked using Primer Map (http://www.bioinformatics.ord/sms2/ pri-mer_map.html). The final reaction mixture contained SYBR® Green Super-mix (Bio-Rad), forward and reverse primer (Thermo Fisher Scientific, Ger-many), cDNA template and MilliQ-water. Each sample was run in tripli-cates. To control for genomic DNA contamination, an on-column DNase treatment was performed during extraction. Before the cDNA synthesis, a genomic DNA wipeout extraction was used; and the primers were designed across two adjacent exons to avoid unspecific amplification of genomic DNA. Each real time PCR plate contained positive internal controls.

Tissue stabilization (Paper III) Deep frozen (-80°C) whole brains were placed in -20°C the day before stabi-lization to reduce a steep temperature gradient. The brains and pituitaries (separated from the brain before freezing) were stabilized with the bench-top Stabilizor T1 (Denator AB, Sweden) according to the manufacturer´s manual (http://www.denator.com). The stabilization process involves a combination of conductive heat transfer and pressure under vacuum to avoid enzymatic degradation (e.g., of peptides) that might occur during freeze-thawing pro-cesses (Segerström et al., 2016). Whole brains were placed in a Maintainor Tissue card (Denator AB, Sweden) and stabilized in “frozen structure pre-serve” mode and thereafter in “fresh structure preserve” mode to ensure suf-ficient treatment. The stabilized brains were thereafter dissected on ice ac-cording to the rat brain atlas of Paxinos and Watson (1997) and stored at -80°C.

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Radioimmunoassay (Paper II-III) For corticosterone measurements trunk blood was collected. Blood samples were centrifuged at room temperature after which the serum was stored at -20°C. Analysis of corticosterone was performed using the commercial Im-munChemTM Double Antibody Corticosterone 125I radioimmunoassay kit for rats and mice (ICN Biomedicals Inc., CA, USA).

The dissected tissues were heated in acetic acid in a 95° water bath for five minutes and thereafter homogenized with a sonicator. After centrifuga-tion, the supernatants were run on ion exchange columns with conditioned with Sephadex gel. The peptides were eluted with pyridine and formic acid containing buffers with varying ion strength. For more information about tissue extraction and purification see Christensson-Nylander et al. (1985). Radioimmunoassays for and dynorphin B (Nylander et al., 1997), MEAP (Nylander et al., 1995) and N/OFQ (Ploj et al., 2000) have preciously been described. Antiserum for the respective peptide was generated in rabbits. Antibody-bound peptides in the dynorphin B and N/OFQ assays were sepa-rated from free peptides by adding goat-anti-rabbit-IgG and normal rabbit serum. For the MEAP assay, separation was performed by adding charcoal suspension. For β-endorphin measurements, commercial β-endorphin radio-immunoassay kits from Phoenix Pharmaceuticals (CA, USA) were used according to manufacturer’s protocol.

In this thesis, the immunoreactive levels measured by radioimmunoassay will only be named levels from here on.

Statistical analysis Statistical analyses were performed using Statistica (Statsoft Inc., OK, USA) version 12 in Papers II and IV and version 13 in Papers I and III and SPSS (IBM, New York, USA) version 22 in Paper I. For the multivariate data analysis (Paper II), SIMCA-P+ 12.0 (Umetrics AB, Umeå, Sweden) was used. The statistically significant level was set to p < 0.05.

Parametrical statistics Factorial analysis of variance (ANOVA) was used to analyze differences in opioid peptide and corticosterone levels (Papers II and III). A repeated measures ANOVA was used to analyze time-related differences in dopamine and the dose-response function of amphetamine self-administration in Paper IV. Post hoc analyses were performed with Bonferroni (Paper II) and Tuk-ey’s HSD (Papers III and IV). To test differences in the PR trials in Paper IV, Student’s t-test was applied.

43

Non-parametrical statistics Kruskal Wallis, together with the Mann-Whitney post hoc test, was used to analyze effects of rearing and ethanol intake, on gene expression in Paper I. To investigate possible interactions between rearing and intake a two-factor analysis was tested using the Univariate General Linear Model test, two-way ANOVA with type III sum of squares. Correlation analyses in paper I were performed using Spearman correlations.

Multivariate analysis In Paper II, the multivariate method partial least square discriminant analysis was used to complemented conventional statistics to illustrate patterns among individual rats and the peptide and corticosterone levels of im-portance for creating such patterns.

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Results and discussion

Early life stress, alcohol intake and opioid gene expression Early life stress targets the endogenous opioid system and affects the gene expression of opioid receptor and ligands in non-classical reward areas i.e., the amygdala and dorsal striatum. The response to ethanol was altered in animals subjected to early life stress as evidenced by an interaction between voluntary ethanol drinking in adulthood and the expression of opioid genes.

Amygdala Animals exposed to early life stress, i.e., 360 minutes of daily maternal sepa-ration, expressed more Pomc in the amygdala than the MS15 and AFR groups (Figure 10A).

Figure 10. Pomc expression in amygdala. A) Effects of rearing conditions B) Inter-action between early life stress and adult ethanol drinking. *p≤0.05; **p≤0.01, (Mann-Whitney U-test). The Univariate General Linear Model test, two-factor ANOVA with Type III sum of squares showed an interaction effect between rearing (maternal separation) and ethanol drinking (MS x Ethanol). #: p ≤ 0.05 (Mann-Whitney U-test) water vs. ethanol drinking MS360 rats. Abbreviations: AFR, animal facility rearing; AU, arbitrary units; EMM, Estimated Marginal Means; MS, mater-nal separation 15/360 minutes; Pomc, proopiomelanocortin; w, water.

AFRw MS15w MS360w

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*

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MS x Ethanol #

EthanolWater

A B

45

Furthermore, ethanol affected expression solely in the MS360 group; the expression of Pomc was lower than the water-drinking MS360 rats and clos-er to the MS15 rats, whose expression of Pomc was unaffected by ethanol drinking (Figure 10B). These results indicate a normalizing effect after etha-nol drinking, i.e., a reduction of the initially high, Pomc expression levels in the MS360 rats.

The product from Pomc expression is proopiomelanocortin, which is cleaved enzymatically into several biologically active peptides (see Endoge-nous opioids and Figure 2); hence, it is difficult to interpret the outcome of the alterations in Pomc expression. The literature regarding ethanol, β-endorphins and the amygdala is scarce but there are some reports that β-endorphin increases in the central amygdala after acute exposure to ethanol (Lam and Gianoulakis, 2011; Lam et al., 2008). The higher levels of Pomc expression could also be due to increased adrenocorticotropic hormone (ACTH) and/or melanocyte-stimulating hormone (MSH). A functional study by Brunson et al. (2001) shows that ACTH can act directly on neurons in the amygdala and downregulate the expression of CRF, which is highly involved in the response to ethanol (Koob, 2008). Furthermore, ethanol exposure dur-ing adolescence and adulthood reduces alpha-MSH in the central amygdala (Lerma-Cabrera et al., 2013; Navarro et al., 2008), an effect that resembles the interaction effect found herein.

Dorsal striatum In the dorsal striatum, the expression of opioid receptors was dependent on rearing conditions (Figure 11).

Figure 11. Effects of rearing conditions on A) Oprd1, B) Oprk1 and C) Oprm1 gene expression in the dorsal striatum. *p≤0.05; **p≤0.01 (Mann-Whitney U-test). Ab-breviations: AFR, animal facility rearing; AU, arbitrary units; MS, maternal separa-tion 15/360 minutes; w, water drinking animals; Oprd1, Opioid receptor delta 1; Oprk1, opioid receptor kappa 1; Oprm1, opioid receptor mu 1.

AFRw MS15w MS360w0

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)

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AFRw MS15w MS360w0

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on (A

U)

*A B C

46

Rats exposed to 360 minutes of maternal separation had higher expression of Oprd1 (Figure 11A) and Oprk1 (Figure 11B) than the AFR and MS15 rats, and higher Oprm1 expression than the AFR (Figure 11C).

Previous studies show that rats exposed to MS360 have lower immunore-active levels of MEAP in the dorsal striatum than MS15 rats (Gustafsson et al., 2008; 2007). Decreased MEAP could be either a cause or an effect of upregulated δ-opioid receptors, i.e., increased expression of Oprd1. The exact role of opioid receptors in the dorsal striatum is not fully understood, but they are regulators of other neurotransmitters such as dopamine, GABA and glutamate (Atwood et al., 2014; Banghart et al., 2015; Christensson-Nylander et al., 1986; Herrera-Marschitz et al., 1986; Steiner and Gerfen, 1998).

Evidence that ethanol affects endogenous opioids in the dorsal striatum can be seen in a series of experiments by Mendez et al., in which they stud-ied the acute impact of forced administration of ethanol. They show that a single administration of ethanol (2.5 g/kg) increased both binding of a δ-receptor ligand and Penk expression 1–2 hours later (Mendez et al., 2004; 2008). Furthermore, increased binding was not found when using a μ-receptor ligand (Mendez et al., 2003). Voluntary drinking shows that high intake increases δ-receptor activity and that intra-striatal injection of naltrin-dole (a selective δ-receptor antagonist) decreases ethanol drinking (Nielsen et al., 2012). However, the literature is inconclusive regarding pharmacolog-ical treatments with δ-receptor antagonists and agonists and the effects on ethanol intake; studies show a decrease, increase or no effect on intake after δ-receptor modulation (Ciccocioppo et al., 2002; Franck et al., 1998; Hyytia and Kiianmaa, 2001; Ingman et al., 2003; Krishnan-Sarin et al., 1995; Margolis et al., 2008).

In Paper I, an interaction between early life stress and ethanol intake was found in the expression of Oprd1 (Figure 12A) and Oprk1 (Figure 12B). The expression of both genes was reduced in ethanol-drinking MS360 rats com-pared to the water controls, whereas ethanol-drinking MS15 rats had similar expression levels as the controls. These data agree with previous findings showing that voluntary drinking results in higher striatal MEAP levels in MS360 rats whereas the MEAP were unaffected by ethanol in the MS15 rats (Gustafsson et al., 2007). Furthermore, MS360 and MS15 rats respond dif-ferently to naltrexone in a voluntary ethanol intake paradigm (Daoura and Nylander, 2011). The lower expression of δ-receptors seen in our study and the previous findings of higher MEAP after ethanol, could contribute to dif-ferences in treatment response.

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Figure 12. Interaction and main effects for early life stress and adult ethanol drink-ing on A) Oprd1 and B) Oprk1 expression in the dorsal striatum. The Univariate General Linear Model test, two-factor ANOVA with Type III sum of squares shows an interaction effect between rearing (maternal separation) and ethanol drinking (MS x Ethanol) and a main effect of ethanol (Ethanol). *p<0.05, ***p<0.001 MS360 water vs. MS 360 ethanol (Mann-Whitney U-test). Abbreviations: EMM, estimated marginal means; MS, maternal separation 15/360; Oprd1, opioid receptor delta 1; Oprk1, opioid receptor kappa 1.

Adolescent housing conditions and opioid peptides Short and prolonged single housing in early adolescence resulted in time-, area- and peptide specific changes in endogenous opioids.

Nociceptin/Orphanin FQ A short period of single housing (30 minutes) increased N/OFQ in the amygdala, nucleus accumbens and medial prefrontal cortex (Figure 13). Furthermore, a trend (p=0.07) of higher levels was seen in the hypothalamus. The animals housed singly for a short period had higher levels in the medial prefrontal cortex and hypothalamus than the rats exposed to a prolonged period of single housing (Figure 13). N/OFQ were unaffected by seven days of single housing (Figure 13). Increased serum corticosterone was seen only after the short-term single housing.

Corticosterone levels are unaffected by a centrally administered NOP re-ceptor antagonist (UFP-101), but the antagonist attenuates the effect of in-tracerebroventricular N/OFQ, which suggests that N/OFQ is not tonically active (Leggett et al., 2006). An acute stress challenge increases Oprl1 (i.e., the gene encoding N/OFQ) expression and CRF in central and basomedial amygdala, and acute stress also increases Oprl1 expression in the para-ventricular hypothalamus whereas chronic stress does not (Ciccocioppo et al., 2014; Green and Devine, 2009). Furthermore, N/OFQ blocks CRF-

MS360MS15

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A B

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MS x EthanolEthanol

MS x EthanolEthanol

******

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induced inhibitory postsynaptic potentials in central and basolateral amygda-la; this inhibition is even greater in restrained rats (Ciccocioppo et al., 2014). Behavioral data regarding the effects of NOP activation are conflicting, as anxiolytic effects are reported for both agonists, and antagonists (Duzzioni et al., 2011; Fernandez et al., 2004; Gavioli et al., 2003; Green et al., 2007; Jenck et al., 2000; Koster et al., 1999; Varty et al., 2005; 2008; Vitale et al., 2009).

Figure 13. The effects of short and prolonged single housing on N/OFQ in different brain areas. The values are presented as a percentage of the levels in the control group. *p<0.05; **p<0.01, compared to group housed controls; #p<0.05: ##p<0.01, compared to single housed rats (one-factor ANOVA followed by Bonferroni post hoc test). Abbreviations: AMY, amygdala; FCx, frontal cortex; HC, hippocampus; HT, hypothalamus; LSH, long single housing; MPFCx, medial prefrontal cortex; NAc, nucleus accumbens; N/OFQ, nociceptin/orphanin FQ; PAG, periaqueductal gray; SSH, short single housing; Sn, substantia nigra; dStr, dorsal striatum; VTA; ventral tegmental area.

A hypothesis in Witkin et al. (2014), suggests that N/OFQ is recruited after acute stress to facilitate adaption to the new environmental condition. When the stressful stimuli are protracted, stimulation of N/OFQ activity at extrahy-pothalamic areas may serve to attenuate the pathological consequences asso-ciated with chronic stress. This hypothesis could explain why the acute stress (i.e., short single housing) increased N/OFQ and circulating corticosterone, but the levels were similar to those of group-housed non-stressed, rats during prolonged single housing.

When stress-induced effects on the N/OFQ system are investigated, the methods used are usually: restraint stress, forced swim test, defeat, chronic unpredictable stress, open field, or elevated plus maze. In Paper II, single housing, a mild, common, and sometimes necessary procedure in experi-mental research, was also able to induce a significant stress response in ado-lescent animals.

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Methionine-Enkephalin-Arginine6-Phenylalanine7

Rats subjected to seven days of single housing in early adolescence had low-er MEAP levels than group-housed animals in the hypothalamus, frontal cortex, medial prefrontal cortex, hippocampus, amygdala, substantia nigra and periaqueductal gray (Figure 14). In addition, reduced hippocampal levels of MEAP were found in short- compared to prolonged single housed rats (Figure 14). Short single housing had no effect on MEAP levels (Figure 14).

Figure 14. The effects of short and prolonged single housing on MEAP in different brain areas. The values are presented as a percentage of the levels in the control group. *p<0.05; **p<0.01, compared to group housed controls; #p<0.05: ##p<0.01, compared to single housed rats (one-factor ANOVA followed by Bonferroni post hoc test). Abbreviations: AMY, amygdala; FCx, frontal cortex; HC, hippocampus; HT, hypothalamus; LSH, long single housing; MEAP, Methionine-enkephalin-Arginine6-Phenylalanine7, MPFCx, medial prefrontal cortex; NAc, nucleus accum-bens; PAG, periaqueductal gray; SSH, short single housing; Sn, substantia nigra; dStr, dorsal striatum; VTA; ventral tegmental area.

The enkephalin system continues to mature after birth and during the first weeks of life δ-receptors and enkephalin levels increase (Bayon et al., 1979; Kornblum et al., 1987; Spain et al., 1985); therefore, this system might be extra sensitive to environmental disturbances. As mentioned in the introduc-tion, social contacts during adolescence are important for normal develop-ment and endogenous opioids are highly involved in mediating the reward-ing effect of this behavior. Deprivation of social contact during this period could result in an increased state of anxiety as reflected by reduced levels of enkephalins.

Behavioral tests with Oprd1 and Penk knockout mice show increased anxiety-like behavior in a variety of tests (Filliol et al., 2000; Konig et al., 1996; Ragnauth et al., 2001). Pharmacological studies with systemic admin-

50

istrations and local injections of δ-receptor agonists into the amygdala, cin-gulate cortex, and hippocampus decrease the anxious behavior (Perrine et al., 2006; Randall-Thompson et al., 2010; Saitoh et al., 2004; 2018; Vergura et al., 2008). Likewise, the administration of antagonists increases the anxie-ty-like behaviors (Narita et al., 2006; Perrine et al., 2006; Saitoh et al., 2004; 2005). Surprisingly however, specific Oprd1 gene inactivation in forebrain GABAergic neurons results in a low-anxiety phenotype (Chu Sin Chung et al., 2015), indicating that there might be a more complex and regions-specific effects of enkephalin signaling and anxiety. Furthermore, it has been shown that enkephalin expression in the basolateral amygdala is linked to stress coping (Berube et al., 2013; 2014) and enkephalins may be implicated in both vulnerability and resilience to stress (Henry et al., 2017). Palm and Nylander (2014a) investigated the effects of 24 hours or six weeks of single housing and showed that MEAP increases in the hippocampus, medial pre-frontal cortex and in the pituitary gland at 24 hours, but a residual increase is only found in the cingulate cortex. The results from Paper II, together with those in Palm and Nylander (2014a) suggest a time-dependent change in the MEAP system after exposure to single housing, with an initial increase after 24 hours. This is followed by declining levels and, after a period of time, the levels reset.

Dynorphin B Dynorphin B was unaffected by either short or prolonged single housing. This was also demonstrated by Palm and Nylander (2014a) where dynorphin B levels remained unchanged 24 hours and six weeks after isolation, with an exception of reduced levels in the amygdala. The maturation of the dy-norphin system occurs at an earlier stage than the enkephalin one, (Kornblum et al., 1987; McDowell and Kitchen, 1987); furthermore, κ-receptors and dynorphin signaling are not as involved as β-endorphin and enkephalins in the age-specific play behavior (Niesink and Vanree, 1989; Vanderschuren et al., 1995b). Therefore, single housing may not affect the dynorphins to the same extent as enkephalins.

Adolescent alcohol exposure Intermittent ethanol exposure during adolescence induced alterations in en-dogenous opioid peptides and dopamine dynamics in brain areas involved in stress regulation, reward, and reinforcement.

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Effects of intoxication on opioid peptides

The nucleus accumbens was the brain area most affected by intoxication. β-endorphin levels were lower than for their time-matched water controls; this effect was short-lived and not present three weeks post-ethanol (Figure 15A). This is in contrast to studies on adult rats where β-endorphin increases in the nucleus accumbens after acute ethanol exposure (Lam et al., 2010; Marinelli et al., 2004; 2003). This could be due to age-related differences, but also differences in the methodology, or acute vs. repeated ethanol expo-sures. As mentioned in “Social behavior and endogenous opioids”, the en-dogenous opioid system is involved in the social play that occurs during adolescence, and activation of μ-receptors in nucleus accumbens is important for mediating the behavior (Manduca et al., 2016; Trezza et al., 2011). Etha-nol interferes with social play behavior (Trezza et al., 2014) and hence, the ethanol could have affected the normal play development, which would ex-plain the differences in β-endorphin levels in the nucleus accumbens in Pa-per III.

Figure 15. β-endorphin (A) and MEAP (B) levels in the nucleus accumbens after repeated ethanol exposure during adolescence. Levels were measured in an ethanol-intoxicated state (two hours after the last exposure) or three weeks after the exposure (residual effects). “Treatment x time” and “Treatment” indicates a significant inter-action effect and an effect of treatment, respectively (*p<0.05 two-factor ANOVA). Statistical between-groups differences were tested with Tukey’s HSD post hoc test (#p<0.05). Abbreviation: MEAP, Methionine-enkephalin-Arginine6-Phenylalanine7.

An interaction between treatment (ethanol or water) and time (two hours or three weeks after the last exposure) was seen in the accumbal levels of MEAP, with the highest levels occurring in the intoxicated state (Figure 15B). The literature suggests that ethanol intoxication increases enkephalins in the nucleus accumbens of adult rats. An increase of Penk expression and δ-receptor binding in both shell and core of the accumbens can be seen two

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hours after ethanol administration (Mendez and Morales-Mulia, 2006; Oliva et al., 2008). Awake rats have increased accumbal levels of Methionine-enkephalin when injected with 1.6 g/kg ethanol. However, higher and lower doses than 1.6 g/kg have no effect on Methionine-enkephalin (Marinelli et al. 2005). In anesthetized rats, 2.5 g/kg leads to a peak of Methionine-enkephalin at 30 minutes whereas lower doses delay the peak (Mendez et al. 2010).

Residual effects on opioid peptides In several brain areas, the effects of ethanol exposure on MEAP persisted three weeks post ethanol (Figure 16).

Figure 16. MEAP levels in (A) amygdala, (B) ventral tegmental area and (C) sub-stantia nigra after repeated ethanol exposure in adolescence. MEAP were measured in an ethanol-intoxicated state (two hours after last exposure) or three weeks after the last exposure (residual effects). “Treatment” indicates a significant effect of treatment, (*p<0.05, **p<0.01 two-factor ANOVA). Abbreviations: EtOH, ethanol; MEAP, Methionine-enkephalin-Arginine6-Phenylalanine7.

MEAP were lower in the amygdala after ethanol exposure (Figure 16A). As mentioned in “Adolescent housing conditions and opioid peptides”, low levels of enkephalins are associated with increased anxiety and stress. Im-portantly, the amygdala is a key region for mediating these emotional states (Janak and Tye, 2015; Tye et al., 2011). In the present thesis, behavioral manifestations after adolescent ethanol exposure were not studied, but other studies have reported increased anxiety-like behaviors (Van Skike et al., 2015; Varlinskaya et al., 2016), which could be a consequence of low enkephalin levels. δ-receptor knockout mice have an increased consumption of ethanol (Roberts et al., 2001) and their elevated ethanol intake may be a way to reduce an innately high anxiety level (Chu Sin Chung and Kieffer, 2013). The low enkephalin after adolescent ethanol exposure may therefore constitute a risk factor for elevated intake of ethanol later in life.

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In the VTA and substantia nigra, MEAP were higher in ethanol-exposed rats (Figure 16B and 16C). In the intoxicated state (two hours), the dy-norphin B levels in the substantia nigra were the same for the ethanol-treated group and water controls, but higher levels were found in the ethanol group at three weeks after the last exposure. The substantia nigra and VTA contain dopaminergic neurons that extend into the striatal, limbic and cortical areas (Björklund and Dunnett, 2007). Importantly, endogenous opioids are highly involved in regulating dopamine output (Christensson-Nylander et al., 1986; Fields and Margolis, 2015; Herrera-Marschitz et al., 1986).

Residual effects on in vivo dopamine in the dorsal striatum Repeated episodic binges of ethanol during adolescence did affect dopamine dynamics in dorsal striatum.

A single amphetamine challenge significantly increased the peak area re-sponse in water controls at 5, 15, 25 and 35 minutes. In the ethanol-exposed animals, the increase was only seen at the 5-minute time point. The peak area involves two parameters—peak amplitude (the amount dopamine re-leased) and T80 (the time of dopamine reuptake). For more information see “In vivo high-speed chronoamperometry – analysis of dopamine”. The dif-ference in peak area was mainly driven by an interaction effect between time and treatment in peak amplitude whereas the T80 was comparable for the two groups (see Figure 17 A and B). Hence, these results indicate that ado-lescent ethanol exposure suppresses dopamine release rather than inducing a more efficient dopamine removal.

Palm and Nylander (2014b) investigated the dopamine response in dorsal striatum after voluntary ethanol intake and showed that ethanol drinking in adolescence reduces basal (i.e., pre-amphetamine challenge) potassium-induced peak amplitudes in adulthood, but the response to a single amphet-amine challenge is unaffected. The discrepancy with our results could be due to methodological differences; Paper IV describes the effects of ethanol binges during adolescence independent of phenotypes, whereas Palm and Nylander (2014b) describes the effects in a drinking phenotype.

The effect of adolescent ethanol exposure in the ventral part of striatum is more studied than the dorsal part. Shnitko and colleagues (2016) did not find any alterations in baseline dopamine release or uptake after electric stimula-tion of VTA in ethanol-exposed rats. Studies using microdialysis show that repeated ethanol exposure increases the basal levels of extracellular dopa-mine in adolescent rats, but these effects do not persist into adulthood (Pascual et al., 2009). An ethanol challenge after a history of adolescent ethanol results in decreased accumbal dopamine release 7 and 14 days after treatment, but at 28 days the effect is diminished (Zandy et al., 2014). In contrast, it has also been reported that adolescent exposure increases extra-cellular dopamine levels after an ethanol challenge (Badanich et al., 2007;

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Pascual et al., 2009; Philpot et al., 2009). Furthermore, adolescent alcohol blunts the acute ethanol effects on evoked dopamine release in nucleus ac-cumbens (Shnitko et al., 2016). Beside the striatum, effects of adolescent ethanol exposure on dopamine dynamics have also been reported in the VTA (Avegno et al., 2016; Schindler et al., 2016) and cortical regions (Trantham-Davidson et al., 2017)

Figure 17. In vivo chronoamperometric recordings in the dorsal striatum. Ampli-tudes (A and C) and T80 (B and D) measurements over time after different drug exposures. The graphs show the potassium-induced dopamine responses after an intravenous amphetamine challenge (2 mg/kg intravenously) as percent (mean± SEM) of reference values after adolescent ethanol or water exposure (A and B) and after adolescent ethanol exposure followed by amphetamine self-administration (C and D). **p<0.01 compared to the time point (−5) before the amphetamine chal-lenge, repeated measures ANOVA followed by Tukey’s HSD post hoc test. Abbre-viations; Amph, amphetamine; SA, self-administration.

Subsequent amphetamine self-administration Although a history of adolescent ethanol exposure increases subsequent vol-untary intake of ethanol in rats (Alaux-Cantin et al., 2013; Amodeo et al., 2017; Criado and Ehlers, 2013; Pascual et al., 2009), the literature is incon-clusive; several studies report only subtle or no effects on subsequent intake

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(Gilpin et al., 2012; Siegmund et al., 2005; Slawecki and Betancourt, 2002; Vetter et al., 2007). Surprisingly, little work has investigated how adolescent ethanol exposure influences intake of drugs other than ethanol. With regard to the growing evidence that adolescent ethanol exposure affects dopamine transmission, drugs that specifically target this system are of interest. Etha-nol exposure during early/mid adolescence sensitizes the rewarding effects of cocaine and attenuates the aversive effects; it also altered gross locomotor activity (Hutchinson and Riley, 2012; Hutchison et al., 2010). However, adolescent ethanol drinking does not alter cocaine self-administration (0.5 and 0.75 mg/kg) in mice on a FR1 schedule of reinforcement (Esteve-Arenys et al., 2017).

As mentioned in “Neuronal circuitries in the addiction cycle”, the dorsal striatum is involved in habitual drug consumption and transition to compul-sive drug use (Everitt and Robbins, 2013). On the basis of the results from the chronoamperometric recordings (see Adolescent ethanol exposure: Re-sidual effects on in vivo dopamine), we hypothesized that there might be a difference in the rats’ motivational drive to consume amphetamine. Howev-er, there were no differences between the water- and ethanol-exposed rats in the PR trials (Figure 18A). A dose-response function was used to explore self-administration behavior on a FR schedule, but the groups displayed similar intake behavior at all doses tested (Figure 18B).

Figure 18. Amphetamine self-administration. A) Progressive ratio trial. Breakpoint values and corresponding response requirements for the last earned reward. The bars represent mean + SEM. B) Dose-response function. The values represent mean (± SEM) number of amphetamine infusions earned on a fixed-ratio three schedule across different unit doses.

Hence, differences in peak area—driven by reduced release of dopamine after amphetamine challenge in the ethanol-exposed group—had no influ-ence on drug-taking behavior during the relatively short period of self-administration tested herein. A different setup with prolonged self-

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administration or a shift toward lower doses could possibly reveal more sub-tle alterations in self-administration behavior.

Subsequent amphetamine self-administration and in vivo dopamine In vivo dopamine dynamics were analyzed in the dorsal striatum two weeks after the last amphetamine self-administration session in rats with and with-out a history of adolescent ethanol exposure. The response to this ampheta-mine challenge was dependent on adolescent history. Ethanol-exposed rats did not increase their peak area after the amphetamine challenge, but the water-exposed animals had an increased peak area at 5, 15, 35 and 45 min. The reduced response was driven by an abolished effect on T80 (Figure 17D) and not due to differences in peak amplitude (Figure 17C) indicative of differences in dopamine transporter function. Even though no differences were noted in amphetamine self-administration behavior, the response to the drug in the brain was altered in ethanol-exposed animals. These results indi-cate a potentially synergistic effect on dopaminergic response to ampheta-mine after adolescent ethanol and adult amphetamine exposure. It would have been interesting to investigate how these rats responded if reintroduced to amphetamine self-administration to for example, another PR trial, lower unit doses of amphetamine or extended repeated periods of self-administration.

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Conclusions

The present thesis provides further evidence that early life stress affects the endogenous opioid system. The expression of four opioid genes—Oprd1, Oprk1, Oprm1, in the dorsal striatum and Pomc in the amygdala—is affected by 360 minutes of maternal separation. Interestingly, changes in gene ex-pression after voluntary ethanol intake depend on early life conditions.

Levels of endogenous opioid peptides are also affected by single housing in early adolescence. A short period of single housing activates the HPA axis and increases N/OFQ in the amygdala, hypothalamus and medial prefrontal cortex. Prolonged single housing causes lower basal levels of MEAP in the amygdala, substantia nigra, periaqueductal gray, hippocampus, hypothala-mus and in frontal- and medial prefrontal cortex. Shorter periods of single housing are common during behavioral measurements, pharmacological treatments, or when terminating an experiment. When measuring individual consumption behavior such as ethanol intake, the animals are often single-housed for longer periods. The knowledge of how endogenous opioid pep-tides are affected by housing conditions has to be taken into consideration when designing an experiment; otherwise, the interpretation of the results would be incorrect.

Repeated ethanol binges during adolescence also affect the endogenous opioid system. The levels of MEAP and β-endorphin in the nucleus accum-bens change during intoxication and residual effects of ethanol exposure are found in MEAP levels in the amygdala, substantia nigra and VTA. The amygdala is involved in the regulation of stress and anxiety, while the sub-stantia nigra and VTA are important for dopaminergic projections in the reward circuitry, which is regulated by endogenous opioids. Furthermore, adolescent ethanol exposure causes alterations in dopamine dynamics in the dorsal striatum; a single amphetamine challenge reduces dopamine release although the reuptake of dopamine remains unaffected. Ethanol-exposed rats and controls display similar amphetamine self-administration behavior, but our analysis shows a more efficient striatal removal of dopamine in ethanol-exposed rats.

It has been postulated that changes in the brain stress system and in do-paminergic activity increase susceptibility for alcohol dependence. The al-terations in endogenous opioids and dopamine dynamics presented in this thesis could partly explain why stress early in life and exposure to ethanol in adolescence increases the risk of elevated alcohol consumption later in life.

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Populärvetenskaplig sammanfattning

Från att du föds, genom hela din ungdomstid och fram till att du befinner dig i mitten av 20-årsåldern så utvecklas och mognar din hjärna. Under den här tiden är hjärnan även extra känslig för olika former av yttre påverkan. Två exempel på faktorer som kan ha negativ effekt på hjärnan är stress och expo-nering av droger. Om du växer upp i en otrygg och stressfylld miljö eller utsätts för upprepad exponering av droger i ung ålder så ökar exempelvis risken att du drabbas av en psykiatrisk sjukdom senare i livet.

Den här avhandlingen har undersökt hur två av hjärnans signalsubstanser, dopamin och endogena opioider (kroppsegna morfinliknande ämnen), på-verkas när en ung individ utsätts för stress eller upprepat intag av alkohol. Dopamin och endogena opioider utgör en viktig del av hjärnans belönings-system och hjälper till att förmedla de positiva känslor som du upplever när du till exempel äter en god middag, har sex eller uppnår en framgång. Dessa positiva känslor kallas för naturlig belöning. Men det är inte bara vid naturlig belöning som aktiviteten av dopamin och endogena opioider ökar, även alla beroendeframkallande droger har den här egenskapen.

För att undersöka hur den unga hjärnan påverkas av droger eller stress an-vänds ofta djurmodeller. Anledningen är att dessa frågeställningar är svåra att besvara i människa på grund av etiska principer. I den här avhandlingen har därför djurmodeller använts för att simulera olika typer av negativa livs-händelser som förknippas med en otrygg uppväxtmiljö. I en av avhandling-ens studier stördes kontakten mellan mamman och ungarna genom tidsbe-gränsade separationer av nyfödda råttungar och i en annan av studierna pla-cerades unga råttor tidvis ensamma för att efterlikna en miljö med störd social kontakt. Alkohol gavs även till unga råttor för att undersöka hur alko-hol påverkar den unga hjärnan.

Resultaten från studierna visade bland annat att en otrygg uppväxtmiljö påverkade endogena opioider i dorsala striatum - en del av hjärnan som bland annat är kopplad till bildandet av vanor. En otrygg uppväxt förändrade även hur de endogena opioiderna svarade på alkoholintag i vuxen ålder. De endogena opioiderna påverkades även av en störd social miljö under ung-domstiden då minskade nivåer sågs i många av hjärnans olika regioner. Kvarvarande förändringar i både endogena opioider och dopamin syntes efter att alkohol intagits under ungdomstiden. Intaget av alkohol i ung ålder hade ingen effekt på viljan att självadministrera amfetamin, däremot skiljde

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sig dopaminaktiviteten åt i dorsala striatum efter en injektion av amfetamin i vuxen ålder.

Otrygghet, stress och alkohol formar med andra ord den unga hjärnan och förändrar dess belöningssystem. Detta kan, till viss del, förklara varför en otrygg uppväxtmiljö och upprepat intag av droger i ung ålder ökar risken för psykiatrisk sjukdom senare i livet.

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Acknowledgment

Now this period of my life is finished, without my main supervisor Ingrid Nylander it would not have been possible. Thank you for believing in me! To know that you have trust in what I do creates a stimulating environment to work in. Even if you have tons of work to do, you always try to find some time to help. I´m very happy to have had you as my main supervisor! My co-supervisors. Anne-Lie Svensson, you are the gate-way to a PhD. Without your fantastic supervision during SOFOSKO projects, many theses would not have been written. You are one of the most positive persons I know! Maria Ellgren, I have missed you a lot since you left the academia, you were a wonderful support when you worked here. Erika Roman, for all the knowledge that you willingly share about animals, without that, this the-sis would be full of caveats. To all the members of the NAB-group, thanks for being great colleagues. Lova, you are a fantastic person, who does so much for others. I don’t know a single person in the department who haven´t been helped by you. You de-serve the best. Stina and Nikita, it was good to have some familiar PhD-faces when coming back to the lab, keep up the good work. Victoria and Lena, for help and support with teaching and students. Åsa, for being an energy injec-tion! Marita, for all the help with animal work. Carin, for taking care of the everyday maintenance of lab stuff. Sam, a lot of fun left the lab when you headed back to England, you ́re missed! Sara and Loudin, thanks for intro-ducing and welcoming me as a new PhD student and for all the travelling to conferences! Shima, for being my light and laughter in the office for the first years. The distance between Norrland and Middle East has never been so small. To all the students who has helped me in my projects. The Comasco-group, Erika, Aniruddah, Megha and Maria, for your hard work and fruitful discussions! The staff in department of Pharmaceutical Biosciences for helping out with everyday problems. Especially to all PhD students, the past ones (Alfhild, Anna, Anneli, Erika, Helen, Inga, Jenny, Jonathan, Mats, Oskar, Patrik) for showing it is possible and to the present ones (Lisa (You go!), Erik, Sofia)

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for doing it. Lena (O ́Raili) for being a reliable source of knowledge, the solid rocks in the department. One “Blaj” a day keeps the doctor away! Shima, thanks for introducing me to the fantastic group of friends you have. That you having fantastic friends is off course no surprise! Emelie, you are wise, fun and helpful, what else can one ask for? Srebrenka, why didn’t we hang out more during the phar-macy program? I’m glad we had a second chance, I love the way you ap-proach life! Sahar, the sweetest and toughest person I know. Sofie, the calm-ness and the (positive) nerdiness of your personality are a perfect match. Maria, your ability of being both superfun an superserious is amazing! Jenny, Alexandra and Mikka, we are having a 12 (!) years anniversary this year. I hope our 2017-babies will share the same friendship. To all the babies of the next generation (Alma, Abbe, Astrid, Evy, Iris, Greta and Sigge) that decided to be born around the time I was on maternal leave. I had so many great parents to hang out with because of you. I hope I can fol-low you through your lives. An extra thank to Abbe, it has been great to have your mom (Elin) to run beside in the last miles of the PhD-marathon. To my family for being the foundation of my wellbeing, you are the best! I could not have asked for a better environment to grew up in, I really hope I can pass it forward. My PhD period would not have gone this smoothly without being raised of the master of organization and the lifestyle without procrastination, i.e., my mom. I have also tried to apply the calmness that my dad always brings to any situation. To know that the two of you always have my back makes it easier to go through life. My sister Erika for always (or at least before kids came into the picture…) having time to talk and analyzing things and for being inspiring in the way that you really loves your job, if every teacher were as you are there would not be any crisis in the Swedish school system! Per, you have been in our family for such a long time that I almost cannot recall the time before, I´m happy you are a part of “the Granholms”. Bastian, the families little whirlwind, your energy will take you far! My grandmother, the most inspiring 92-year old I know, if I will be half as you are in your age I will be happy. I love you all! My own little family that I love so much. Tobias, for always letting me be me, for spoiling me with great food every day, letting me whine as much as I want, for always remembering me of the funny things in life and making me forget work as soon as I come home. Ebbe, my funniest person in the world, for you being you is the best thing I know. To see you grow up is a privilege, but please can we slow down the time for a while now!

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